Cellular compositions and methods of making and using them

ABSTRACT

The invention relates to cellular compositions comprising hematopoietic cells with the potential or increased potential to form non-hematopoietic cells; methods for producing such cellular compositions; methods for differentiation of cells of cellular compositions of the invention into cells that exhibit morphological, physiological, functional, and/or immunological features of non-hematopoietic cells; and uses of the cellular compositions. The invention also relates to a method for the expansion of hematopoietic stem and progenitor cells.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 10/499,849,filed Nov. 23, 2004, which is a 371 of PCT/CA02/01979, filed Dec. 20,2002, which claims the benefit of U.S. Provisional Patent ApplicationNo. 60/342,586, filed Dec. 21, 2001. Each of these applications isincorporated by reference.

FIELD OF THE INVENTION

The invention relates to cellular compositions comprising hematopoieticcells with the potential or increased potential to formnon-hematopoietic cells; methods for producing such cellularcompositions; methods for differentiation of cells of cellularcompositions of the invention into cells that exhibit morphological,physiological, functional, and/or immunological features ofnon-hematopoietic cells; and uses of the cellular compositions. Theinvention also relates to a method for the expansion of hematopoieticstem and progenitor cells.

BACKGROUND OF THE INVENTION

Organ transplantation has been successfully used to replace or repairdamaged tissues. However, transplantation is limited by the availabilityof donors, and the high costs and radical nature of the surgery. It isevident that alternative procedures to transplantation are desirable.

The grafting of healthy cells into diseased tissue has been proposed asan alternative to organ transplantation. However, the success of suchgrafts is dependent upon the developmental stage of the injected cells.Adult cells generally do not incorporate into tissue but early stageembryonic cells stably integrate. Embryonic cell grafts are notpreferred due to the ethical issues involved, and technical andavailability limitations. Thus, there is a need for alternative sourcesof cells capable of integration into tissues. In particular, a needexists for cell preparations containing cells of various tissues fortransplantation in which (1) the preparation is accepted by the patient,thus avoiding the difficulties associated with immunosuppression, (2)the preparation is safe and effective, thus justifying the cost andeffort associated with treatment, and (3) the preparation is efficaciousduring and after transplantation.

Bone marrow transplantation is a common form of therapy for a number ofdiseases involving dysfunction of hematopoietic cells, or which involvetreatments which irreversibly damage hematopoietic cells (e.g.chemotherapy and radiotherapy for cancer). The use of bone marrowtransplantation has allowed more intensive and effective chemotherapyand radiotherapy for cancer. However, the approach requires an adequatenumber of stem cells to ensure success. Thus, there is a need forsources of hematopoietic stem cells that will reduce the risk of graftversus host rejection and provide an adequate number of stem cells fortransplantation.

The citation of any reference herein is not an admission that suchreference is available as prior art to the instant invention.

SUMMARY OF THE INVENTION

The present inventors have identified distinct cells that candifferentiate into cells of multiple tissue types in vivo and in vitro.The cells were produced by growing hematopoietic cells derived fromumbilical cord blood under selected proliferation conditions. The cellshave properties similar to embryonic stem cells.

The inventors have also developed a method for the expansion ofhematopoietic stem and progenitor cells from umbilical cord blood thatprovides a significant increase in the number of hematopoietic stemcells and progenitor cells available for transplant from a singleumbilical cord. A single umbilical cord yields enough stem cells for onebone marrow transplant, typically for a pediatric patient. In vitroexpansion of the stem cells will increase the possible uses for a singlecord blood collection. Stem cell expansion will allow greateraccessibility to this form of treatment and allow for the development ofcord blood stem cells for gene therapy. In addition, the degree of HLAincompatibility that can be tolerated is greater with cord blood thanwith bone marrow (1). This is important to the establishment of cordblood banks because it increases the donor pool.

Thus, an aspect of the present invention is directed to methods ofproducing cells with potential or increased potential to form differenttypes of non-hematopoietic cells. In accordance with the presentinvention, the method begins with obtaining hematopoietic cells (e.g.from umbilical cord blood) and enriching the cells for hematopoieticstem cells and progenitor cells by positive or negative selection. Theresulting cell preparation enriched for hematopoietic stem cells andprogenitor cells is cultured under proliferation conditions to producecells with potential or an increased potential to form different typesof non-hematopoietic cells. This novel process leads to the preparationof hematopoietic cells with the potential or increased potential to formdifferent types of non-hematopoietic cells in vitro and in vivo.

In an embodiment, a method is provided for converting hematopoieticcells into cells with potential or increased potential to form differenttypes of non-hematopoietic cells. The method of converting hematopoieticcells into newly created cells with potential or increased potential toform non-hematopoietic cells involves obtaining hematopoietic cells(e.g. from umbilical cord blood) and enriching the cells forhematopoietic stem cells and progenitor cells by positive or negativeselection; and culturing the resulting cell preparation underproliferation conditions so that cells in the preparation develop thepotential or an increased potential to form different types ofnon-hematopoietic cells and tissues.

Another aspect of the invention is an enriched hematopoietic cellpreparation that is enriched for hematopoietic stem cells and progenitorcells that are capable of forming cells that have the potential orincreased potential to form cells of multiple tissue types both in vitroand in vivo. In an embodiment, the enriched hematopoietic cellpreparation comprises essentially CD45⁺HLA-ABC⁺ cells (HLA-Class 1+),preferably CD45⁺HLA-ABC⁺Lin⁻.

In another aspect the invention relates to an isolated cellularcomposition comprising or comprising essentially cells with thepotential or increased potential to form hematopoietic andnon-hematopoietic cells in vitro and in vivo. The cells in thecomposition may have an altered differentiation program enabling thecells to form non-hematopoietic cells. The cells have the potential todifferentiate into cells that exhibit morphological, physiological,functional, and/or immunological features of non-hematopoietic cells.The cells may be further characterized by embryonic or earlynon-hematopoietic tissue markers (e.g. early muscle marker Desmin).

In an embodiment, a cellular composition is provided comprisingessentially cells with the potential or increased potential to formdifferent types of non-hematopoietic cells produced by a method of theinvention.

In a particular aspect of the invention, a cellular composition isprovided which is produced by culturing hematopoietic cells comprisinghematopoietic stem cells and progenitor cells, preferably an enrichedhematopoietic cell preparation comprising CD45⁺HLA-ABC⁺ cells, morepreferably comprising CD45⁺HLA-ABC⁺Lin⁻ cells, under proliferationconditions and isolating cells in the culture that have the potential orincreased potential to form different types of non-hematopoietic cellsand hematopoietic cells both in vitro and in vivo.

Cells with the potential or increased potential to formnon-hematopoietic cells in a cellular composition may be induced todifferentiate into cells and tissues of different types ofnon-hematopoietic lineages in vitro or in vivo. Thus, the inventionrelates to methods of isolating a population of essentiallynon-hematopoietic cells from hematopoietic cells with the potential orincreased potential to form non-hematopoietic cells produced by a methodof the invention.

The invention therefore also relates to a purified cellular compositioncomprising or comprising essentially cells with potential or increasedpotential to form different types of non-hematopoietic cells that havebeen induced to differentiate into cells of non-hematopoietic celllineages, preferably cells that exhibit morphological, physiological,functional, and/or immunological features of non-hematopoietic cells. Adifferentiated cell preparation is characterized by expression ofgenetic markers of non-hematopoietic cell lineages (e.g. markers formuscle, neural, adipocyte, osteoclast, osteoblast, endothelial,astrocytes, renal, retinal, cornea, and hepatocyte lineages).

In an embodiment, a cellular composition is provided comprising cellswith the potential or increased potential to form non-hematopoieticcells produced by a method of the invention, in combination with aneffective amount of at least one differentiation factor.

In another embodiment, a cellular composition is provided comprisingmitotic or differentiated cells that are progeny of cells with thepotential or increased potential to form non-hematopoietic cellsproduced by a method of the invention.

In an aspect the invention provides a culture system comprising cells,cell preparations, and cellular compositions of the invention.

The invention also contemplates cells, cell preparations, and cellularcompositions of the invention in combination with a substrate or matrix,preferably a substrate or matrix adapted for transplantation into apatient. The substrate may be an engineered biomaterial or porous tissueculture insert.

The present invention also provides a method for expanding, preferablyselectively expanding, hematopoietic stem cells and progenitor cellsfrom umbilical cord blood. The method comprises (a) culturing anenriched hematopoietic cell preparation from umbilical cord bloodcomprising hematopoietic stem cells and progenitor cells underproliferation conditions; and (b) isolating increased numbers ofhematopoietic stem cells and progenitor cells. “Increased numbers ofhematopoietic stem cells and progenitor cells”, refers to an increase inthe number of cells by at least about 2-fold relative to the number ofhematopoietic stem cells and progenitor cells that are present in aparallel control culture of cells that are not subjected to the sameproliferation conditions. The invention also relates to an expandedhematopoietic stem cell and progenitor cell preparation obtained by thismethod. The term “expanding” or “expansion” contemplates theproliferation of the hematopoietic cells.

In an embodiment the invention provides a method for expandinghematopoietic stem cells and progenitor cells comprising (a) obtainingumbilical cord blood and enriching for hematopoietic stem cells andprogenitor cells by positive or negative selection, preferably enrichingfor CD45⁺HLA-ABC⁺ cells, more preferably CD45⁺HLA-ABC⁺Lin⁻ cells; (b)culturing the resulting enriched hematopoietic cell preparation underproliferation conditions; and (c) isolating increased numbers ofhematopoietic stem cells and progenitor cells.

In an aspect the invention provides a method of identifying the presenceof cells of cell preparations and cellular compositions of the inventionin a mixed cell population comprising: exposing the cell population toan antibody or fragment thereof immunogenetically specific for a markerof such cells, the occurrence of the markers being indicative of thepresence of the cells in the cell population. The antibody may comprisea detectable label. The method may involve a selection step involvingfluorescence-activated cell sorting or magnetic bead separation. In anembodiment, the mixed cell population is contacted with one, two, three,four, five, six, seven, eight, nine, ten, or eleven or more, preferablyall, of the following markers CD45, HLA-ABC, stem cell factor receptor,Flt3ligand receptor, Fgf receptor, an embryonic stem cell protein suchas Oct4, Stage Specific Embryonic Antigen-3 (SSEA3), and/or StageSpecific EmbryonicAntigen-4 (SSEA4), HoxB4, Flk-1, CD34, and CD38. Thismethod of identifying cells may have diagnostic applications in diseasesor disorders involving cells of the present invention. The method may beemployed in the diagnosis of early childhood cancers, stem-cell basedcancers, and endogenous stem-cell assessment in patients. The method mayalso be used to monitor a therapy for diseases or disorders associatedwith or involving cells of the present invention.

Cells, cell preparations, and cellular compositions of the invention canbe used in a variety of methods (e.g. transplantation or grafting) andthey have numerous uses in the field of medicine. Cells with thepotential or increased potential to form non-hematopoietic cells, orcells differentiated there from may be used for the replacement of bodytissues, organs, components or structures which are missing or damageddue to trauma, age, metabolic or toxic injury, disease, idiopathic loss,or any other cause.

In an aspect of the invention, the newly created cellular compositionscomprising cells with potential or increased potential to formnon-hematopoietic cells, or non-hematopoietic cells differentiatedtherefrom, can be used in both cell therapies and gene therapies aimedat alleviating disorders and diseases involving the non-hematopoieticcells. The invention obviates the need for human tissue to be used invarious medical and research applications.

The invention also provides a method of treating a patient with acondition involving a non-hematopoietic cell, in particular a defect ina non-hematopoietic cell, comprising transferring or administering aneffective amount of a cellular composition comprising cells with thepotential to form the non-hematopoietic cells into the patient, whereinthe cells differentiate into the non-hematopoietic cells.

The invention also contemplates a cell line comprising cells with thepotential or increased potential to form non-hematopoietic cellsproduced by a method of the invention that have the ability to migrateand localize to specific regions in a patient where they differentiateinto non-hematopoietic cells typical of the region and they integrateinto the tissue in a characteristic tissue pattern.

Expanded hematopoietic stem cell and progenitor cell preparations of theinvention may be used in both cell therapies and gene therapies aimed atalleviating disorders and diseases involving hematopoietic cells. Theinvention contemplates a method of treating a patient with a conditioninvolving hematopoietic cells comprising transferring to a patient aneffective amount of a cell preparation of the invention comprisinghematopoietic stem cells and progenitor cells.

Cells with the potential or increased potential to formnon-hematopoietic cells may be used to screen for potential therapeuticsthat modulate development or activity of such cells or cellsdifferentiated therefrom.

The cells, cell preparations, and cellular compositions of the inventionmay be used as immunogens that are administered to a heterologousrecipient. The cells, cell preparations, and cellular compositions ofthe invention may be used to prepare model systems of disease. Thecells, cell preparations, and cellular compositions of the invention canalso be used to produce growth factors, hormones, etc.

The invention also contemplates a pharmaceutical composition comprisingcells, a cell preparation, or cellular composition of the invention, anda pharmaceutically acceptable carrier, excipient, or diluent. Apharmaceutical composition may include a targeting agent to target cellsto particular tissues or organs.

The invention provides a method for obtaining non-hematopoietic cellsfor autologous transplantation from a patient's own hematopoietic cellscomprising (a) obtaining a sample comprising hematopoietic cells fromthe patient, preferably from fresh or cryopreserved umbilical cordblood; (b) separating out an enriched cell preparation comprisinghematopoietic stem cells and hematopoietic progenitor cells, preferablyCD45⁺HLA-ABC⁺ cells, more preferably CD45⁺HLA⁻ABC⁺Lin⁻ cells; (b)culturing the cells under proliferation conditions to produce a cellularcomposition comprising cells with the potential or increased potentialto form non-hematopoietic cells.

The invention also relates to a method for conducting a regenerativemedicine business. Still further the invention relates to a method forconducting a stem cell business involving identifying agents whichaffect the proliferation, differentiation, function, or survival ofcells that have the potential to form hematopoietic andnon-hematopoietic cells of the invention. An identified agent(s) can beformulated as a pharmaceutical preparation, and manufactured, marketed,and distributed for sale.

In another aspect, the invention contemplates methods for influencingthe proliferation, differentiation, or survival of cells that have thepotential to form hematopoietic and non-hematopoietic cells bycontacting cells of a cellular composition of the invention with anagent or agents identified by a method of the invention.

The invention also contemplates a method of treating a patientcomprising administering an effective amount of an agent-identified inaccordance with a method of the invention to a patient with a disorderaffecting the proliferation, differentiation, function, or survival ofhematopoietic or non-hematopoietic cells.

The invention also contemplates a method for conducting a drug discoverybusiness comprising identifying factors or agents that influence theproliferation, differentiation, function, or survival of cells that havethe potential to form hematopoietic and non-hematopoietic cells of theinvention, and licensing the rights for further development.

The invention further contemplates a method of providing drugdevelopment wherein a cellular composition of the invention or mitoticor differentiated progeny thereof are used as a source of biologicalcomponents of non-hematopoietic or hematopoietic cells in which one ormore of these biological components are the targets of the drugs thatare being developed.

The invention also relates to methods of providing a bioassay.

In an aspect, the invention features a kit including cells generatedusing a method of the invention, or a mitotic or differentiated cellthat is the progeny of the cells.

The invention is also directed to a kit for transplantation ofnon-hematopoietic cells comprising a flask with medium and cells, a cellpreparation, or a cellular composition of the invention.

The invention also relates to a method of using the cellularcompositions in rational drug design.

In an aspect, the invention relates to a kit for rational drug designcomprising non-hematopoietic cells obtained by a process of theinvention. In an embodiment, the kit comprises hepatocytes andinstructions for their use in toxicity assays. In another embodiment,the kit comprises intestinal cells and instructions for their use in anabsorption assay.

Still another aspect of the invention is a kit for producing cellularcompositions comprising cells that have the potential or increasedpotential to form cells capable of differentiating into cells ofmultiple tissue types both in vitro and in vivo, or for producing anexpanded hematopoietic stem cell and progenitor cell preparation.

DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows the growth and maintenance of Lin− stem cells. There was anincrease in Lin− stem cells after 7 days growth with different growthfactors. Lin− cells were grown in serum free medium with combinations ofFGF-4, SCF and Flt-3 ligand. The best growth and maintenance of stemcells occurs when all three growth factors are present.

FIG. 2 shows that the Lin⁻ stem cells are HLA-ABC⁺ and CD45. Lin⁻ cellswere selected and analyzed by flow cytometry for CD45 and HLA-ABCpositive cells. Day 0 Lin− cells and the same cells grown for 7 days are100% CD45⁺ and HLA-ABC⁺. Day 0 Lin− cells contain two populations ofCD45⁺/HLA-ABC⁺ cells with one expressing lower levels, but still clearlypositive.

FIG. 3 shows the presence of human stromal cells in engrafted NOD/SCIDmice. CD45⁻/HLA-ABC⁺ cells were isolated from bone marrow aspirates fromNOD/SCID mice engrafted with FGF, SCF, FLT-3 ligand cells or day 0 Lin−cells. These cells may be stromal-like cells. This supports theobservation of stromal/mesenchymal cells in the cultures.

FIG. 4 shows the changes in cell population with time in culture.Initial increases in CD34⁺ cells occur in the first 2-3 weeks of growthbut then decline. CD33⁺ cells increase rapidly suggesting that existingcells begin to express this marker. A high proportion of cells is CD45⁺.

FIG. 5 shows the results for transplanting NOD/SCID mice with cellsgrown in Fgf, SCF, FLT3 ligand for 8 days. Expansion of the cells is 3×input. Transplanting the same number of cells per mouse resulted incomparable levels of engraftment.

FIG. 6 shows osteoclast cells. Lin− cells grown in Fgf, SCF, FLT3 ligandfor 4-28 days produced osteoclast cells as determined by A) TRAPpositive staining and B) resorption of a calcium citrate substrate inthe presence of serum and GM-CSF.

FIG. 7 shows Desmin positive cells. Lin⁻ day 0 cells are negative forthe early muscle marker, Desmin. Lin⁻ cells grown for 7 days becomedesmin positive as determined by PCR. These cells remain negative formature muscle markers.

FIG. 8 shows muscle actin (bottom panel) and Myo-D (top panel) positivecells. Lin⁻ cells grown in Fgf, SCF, FLT3 ligand for 7 days then grownunder conditions that support muscle cell growth resulted in cellspositive for Muscle specific actin and Myo-D. Fewer cells are positivefor the mature muscle marker Myo-D.

FIG. 9 shows endothelial cells. Lin− cells are positive for Flk-1 aftergrowth in Fgf, SCF, FLT3 ligand conditions. When these cells are grownin the presence of VEGF the cells elongate and lose Flk-1 as expectedfor endothelial cell development (A). The same cells placed in a matrixwill grow into a network of vessel like structures B-F).

FIG. 10 shows CD31 positive endothelial cells. High numbers of CD31positive cells can be obtained from UCB Lin− cells. Cells were grown inconditions described herein and stained using an IgG control (A), and ananti-CD31 antibody (B and C).

FIG. 11 shows hepatocytes from UCB cells. CYP1A2 positive hepatocyteswere found in the livers of NOD/SCID mice engrafted with human UCB Lin⁻cells. Human, non-blood cells (CD45⁻/HLA⁺) were isolated from engraftedmouse livers by flow cytometry (A). Both CD45⁺/HLA⁺ human blood cellsand the human, non-blood cells were tested for the presence of CYP1A2(B&C). CYP1A2 positive cells represent a functional hepatocyte. Thesecells are fewer in number than the total number of human, non-bloodcells found in the liver. None of the mouse cells or the human bloodcells express CYP1A2.

FIG. 12 shows the immunohistochemistry of engrafted mouse livers.Immunohistochemistry on liver sections with CYP1A2 antibody was done.Positive cells were detected.

FIG. 13 shows astrocytes detected in the Lin⁻ population. Lin⁻ cells arenegative for the astrocyte marker GFAP unless first grown in Fgf, SCF,FL3 ligand. PCR was used to detect the presence of GFAP mRNA.

FIG. 14 shows neural positive cells. Lin⁻ cells grown for 7-14 days arepositive for nestin mRNA by PCR.

FIG. 15 shows the immunocytochemistry of neural cells. Lin⁻ cellspre-grown in Fgf, SCF and Flt3 ligand then placed into DME+ serum arepositive for neurofilament and Parkin. The addition of Retinoic acid(RA) results in first the formation of neurospheres and with furtherculturing, neurofilament positive cells. RA kills off the non-neuralcells in the culture.

FIG. 16 shows adipocyte positive cells. Under the same conditions thatresult in osteoclast growth, adipocytes can be detected. Sudan IV stainwas used to detect adipocyte cells in the cell cultures.

FIG. 17 Lin⁻ cells were grown in FGF, SCF, FLT3 ligand for 0, 4 or 8days. Cell proliferation of ˜3× occurred over the 8 days. 500cells/assay were used and colonies counted after 16 days. There is nosignificant difference between the three groups. This indicated that theexpanded cells are equivalent to the untreated population.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See for example, Sambrook, Fritsch, & Maniatis(2); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glovered. 1985) (3); Oligonucleotide Synthesis (M. J. Gait ed. 1984) (4);Nucleic Acid Hybridization B. D. Hames & S. J. Higgins eds. (1985) (5);Transcription and Translation B. D. Hames & S. J. Higgins eds. (1984)(6); Animal Cell Culture R. I. Freshney, ed. (1986) (7); ImmobilizedCells and enzymes IRL Press, (1986) (8); and B. Perbal, A PracticalGuide to Molecular Cloning (1984) (9). The invention may also employstandard methods in immunology known in the art such as described inStites et al. (10); and Mishell and Shigi (11).

For convenience, certain terms employed in the specification and claimsare collected here.

“Patient” refers to an animal preferably a human, to whom treatment,including prophylactic treatment, with the cells, preparations, andcompositions of the present invention, is provided. For treatment ofthose conditions or disease states that are specific for a specificanimal such as a human patient, the term refers to that specific animal.A “donor” refers to an individual (animal including a human) who orwhich donates hematopoietic cells, in particular umbilical cord bloodfor use in a patient.

“Effective amount” refers to concentrations of components such as growthfactors, cells, preparations, or compositions effective for producing anintended result including proliferation of hematopoietic stem andprogenitor cells, or treating a disease or condition with cells,preparations, and compositions of the invention, or for effecting atransplantation of cells within a patient to be treated.

The terms “administering” or “administration” refers to the process bywhich cells, preparations, or compositions of the invention aredelivered to a patient for treatment purposes: Cells, preparations, orcompositions may be administered a number of ways including parenteral(e.g. intravenous and intraarterial as well as other appropriateparenteral routes), oral subcutaneous, inhalation, or transdermal.Cells, preparations, and compositions of the invention are administeredin accordance with good medical practices taking into account thepatient's clinical condition, the site and method of administration,dosage, patient age, sex, body weight, and other factors known tophysicians.

“Transplanting”, “transplantation”, “grafting” and “graft” are used todescribe the process by which cells, preparations, and compositions ofthe invention are delivered to the site within the patient where thecells are intended to exhibit a favorable effect, such as repairingdamage to a patient's tissues, treating a disease, injury or trauma, orgenetic damage or environmental insult to an organ or tissue caused by,for example an accident or other activity. Cells, preparations, andcompositions may also be delivered in a remote area of the body by anymode of administration relying on cellular migration to the appropriatearea in the body to effect transplantation.

“Essentially” refers to a population of cells or a method which is atleast 20+%, 30+%, 40+%, 50+%, 60+%, 70+%, 80+%, 85+%, 90+%, or 95+%effective, more preferably at least 98+% effective, most preferably 99+%effective. Therefore, a method that enriches for a given cellpopulation, enriches at least about 20+%, 30+%, 40+%, 50+%, 60+%, 70+%,80%, 85%, 90%, or 95% of the targeted cell population, most preferablyat least about 98% of the cell population, most preferably about 99% ofthe cell population. In certain embodiments the cells in an enrichedhematopoietic cell population of the invention comprise essentiallyCD45⁺HLA-ABC⁺ cells, or preferably CD45⁺HLA⁻ABC⁺Lin⁻ cells. In otherembodiments, a cellular composition of the invention comprisesessentially cells with the potential or increased potential to formnon-hematopoietic cells.

“Isolated” or “purified” refers to altered “by the hand of man” from thenatural state i.e. anything that occurs in nature is defined as isolatedwhen it has been removed from its original environment, or both. In anaspect, a population or composition of cells is substantially free ofcells and materials with which it may be associated in nature. Bysubstantially free or substantially purified is meant at least 50% ofthe population are the target cells, preferably at least 70%, morepreferably-at least 80%, and even more preferably at least 90% are freeof other cells. Purity of a population or composition of cells can beassessed by appropriate methods that are well known in the art.

The “fibroblast growth factor receptor” or “FGF receptor” or “FGF-R”refers to proteins that bind to a family of related growth factorligands, the fibroblast growth factor (FGF) family. The term includesthe four FGF transmembrane protein tyrosine kinases, (12, 50), andvariants thereof which may be cell bound or secreted forms. FGFR1 andFGFR2 bind acidic FGF/FGF1 and basic FGF/FGF2 with similar affinity(13). FGFRs bind FGF1 and FGF4 (hst/kfgf) with moderate to highaffinity, while FGFR3 binds to only FGF1 and FGF4 (14, 15). The termalso encompasses FGFR6, FGFR16, FGFR17, FGFR18, and FGFR19. See Moroni Eet al (51) and Goldfarb M. (52) describing fibroblast growth factors andtheir receptors.

The “flt3 receptor” or “flt3” refers to proteins belonging to a familyof structurally related tyrosine kinase receptors that contain fiveextracellular immunoglobulin (Ig)-like domains and an intracellulartyrosine kinase domain (Small et al., Proc. Natl. Acad. Sci. 91:459-463(1994)). See Gilliland D G and Griffin J D. (53) for a review of FLT3.

The stem cell factor (SCF) receptor [synonyms: CD117 protein, SCFreceptor or c-kit receptor (17)], is localized in the plasma membrane ofblood stem cells and is encoded by the proto-onkogen c-kit (18). SeeSmith M A et al (54) for a review of stem cell factor.

“Gene therapy” refers to the transfer and stable insertion of newgenetic information into cells for the therapeutic treatment of diseasesor disorders. A foreign gene is transferred into a cell thatproliferates to introduce the transferred gene throughout the cellpopulation. Therefore, cells and compositions of the invention may bethe target of gene transfer, since they will produce various lineageswhich will potentially express the foreign gene.

As used herein, “hematopoietic cells” refers to cells that are relatedto the production of blood cells, including cells of the lymphoid,myeloid and erythroid lineages. Exemplary hematopoietic cells includehematopoietic stem cells, primordial stem cells, early progenitor cells,CD34⁺ cells, early lineage cells of the mesenchymal, myeloid, lymphoidand erythroid lineages, bone marrow cells, blood cells, umbilical cordblood cells, stromal cells, and other hematopoietic precursor cells thatare known to those of ordinary skill in the art. The hematopoietic cellsmay be obtained from fresh blood, reconstituted cryopreserved blood, orfresh or reconstituted fractions thereof.

The hematopoietic cells (and the cells in the preparations andcompositions of the invention) are preferably mammalian cells; morepreferably, the cells are primate, pig, rabbit, dog, or rodent (e.g. rator mouse) in origin. Most preferably, the cells are human in origin. Thehematopoietic cells may be obtained from a fetus, a child, anadolescent, or an adult.

The most desirable source of the hematopoietic cells is umbilical cordblood (UCB). “Umbilical cord blood” generally refers to blood obtainedfrom a neonate or fetus. In a preferred embodiment, umbilical cord bloodrefers to blood obtained form the umbilical cord or placenta ofnewborns. Hematopoietic cells obtained from UCB offer several advantagesincluding less invasive collection and less severe graft versus host(GVH) reaction (19). The use of umbilical cord blood also eliminates theuse of human embryos as a source of embryonic stem cells. Cord blood maybe obtained by direct drainage from the cord and/or by needle aspirationfrom the delivered placenta at the root and at distended veins.

“Non-hematopoietic cells” include non-blood and non-lymph cells,including but not limited to muscle cells, neural cells, adipocytes,osteoclasts, osteoblasts, endothelial cells, astrocytes, pancreaticcells (e.g. exocrine or endocrine pancreatic cells), retinal cells,renal cells, connective tissue cells, corneal cells, and hepatocytes.

“Cells with the potential or increased potential to formnon-hematopoietic cells” refers to cells, preferably hematopoieticcells, that show at least one phenotypic characteristic of an earlystage non-hematopoietic cell (e.g. stem, precursor, or progenitornon-hematopoietic cells), and preferably at least one phenotypiccharacteristic of an embryonic stem cell. Such phenotypic characteristicscan include expression of one or more proteins specific for early stagenon-hematopoietic cells, or a physiological, morphological,immunological, or functional characteristic specific for an early stagenon-hematopoietic cell or embryonic stem cell [e.g. Oct4, Stage SpecificEmbryonic Antigen-3 (SSEA3), and/or Stage Specific Embryonic Antigen-4(SSEA4)].

Cells with potential or increase potential to form non-hematopoieticcells are produced by first obtaining hematopoietic cells and enrichingthe cells for hematopoietic stem cells and progenitor cells (sometimesreferred to herein as “enriched hematopoietic cell preparation”). Theterm “stem cells” refers to undifferentiated cells that are capable ofessentially unlimited propagation either in vitro, in vivo or ex vivoand capable of differentiation to other cell types. “Progenitor cells”are cells that are derived from stem cells by differentiation and arecapable of further differentiation to more mature cell types. Negativeand positive selection methods known in the ant can be used forenrichment of the hematopoietic cells. For example, cells can be sortedbased on cell surface antigens using a fluorescence activated cellsorter, or magnetic beads which bind cells with certain cell surfaceantigens (e.g. CD45). Negative selection columns can be used to removecells expressing lineage specific surface antigens.

In an aspect of the invention, an enriched hematopoietic cellpreparation is provided wherein the cells in the preparation arecharacterized as follows:

(a) CD45⁺HLA-ABC⁺

(b) Lin⁻;

(c) stem cell factor receptor+

(d) FLT3 ligand receptor+;

(e) FGF receptor+;

(f) CD34⁺;

(g) CD38⁺; and

(h) CD33⁺.

In an embodiment, an enriched hematopoietic cell preparation is providedcomprising cells characterized by (a) and (b); or (a), (c), (d), and(e), and optionally (b), (f), (a), and/or (h).

An enriched hematopoietic cell preparation may comprise cells that areat least 70%, 80%, 90%, 95%, 98%, or 99% CD45⁺HLA-ABC⁺Lin⁻ cells, 70%,80%, 90%, 95%, 98%, or 99% stem cell factor receptor+, 70%, 80%, 90%,95%, 98%, or 99% Flt3ligand receptor+, 70%, 80%, 90%, 95%, 98%, or 99%FGF receptor+, and it may optionally comprise at least 50-80% CD34⁺cells, at least 50-80% CD38⁺ cells, and/or at least 50% CD33⁺ cells.

In an embodiment, an enriched cell population of the invention isprovided comprising the following:

(a) at least 50% CD34⁺ cells, preferably 60 to 95%, more preferably 65%to 90%, or most preferably about 65% CD34⁺ cells;

(b) about 5 to 50%, preferably 5 to 25%, more preferably 5 to 15%, mostpreferably about 10% of the cells in (a) are CD33⁻ and CD38⁻;

(c) at least 50% CD34″; preferably 15 to 40%, more preferably 15% to40%, or most preferably about 35% CD34⁺ cells;

(d) about 5 to 50%, preferably 5 to 25%, more preferably 5 to 15%, mostpreferably about 10% of the cells in (c) are CD33⁺ or CD38⁺ and theremaining cells are negative for all hematopoietic cell surfaceantigens;

(e) about 5 to 50%, preferably 5 to 25%, more preferably 5 to 20%, mostpreferably about 5% are CD33⁺; and

(f) about 20 to 60%, preferably 25 to 55%, more preferably 35 to 45%,most preferably 40% are CD38⁺.

The enriched hematopoietic cell preparation can be cultured underproliferation conditions to produce cells that have potential orincreased potential to form different types of non-hematopoietic cellsand tissues. The enriched preparation of hematopoietic stem cells andprogenitor cells may be cultured in vitro or in vivo, preferably invitro. The proliferation conditions are those conditions that give riseto cells that have the potential or increased potential to formnon-hematopoietic cells and tissues.

The proliferation conditions involve culturing the cells in the presenceof one or more positive growth factors for a sufficient time to enablethe cells to complete sufficient cell cycles to develop tissue potentialor increased tissue potential. Positive growth factors are growthfactors that promote and maintain cell proliferation. Growth factorssuch as TGFβ and TNFα that promote differentiation are not suitable foruse in the proliferation conditions of the method of the invention.

The positive growth factors maybe human in origin, or may be derivedfrom other mammalian species when active on human cells. The followingare representative examples of positive growth factors which may beemployed in the present invention: all members of the fibroblast growthfactor (FGF) family including FGF-4 and FGF-2, epidermal growth factor(EGF), stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand,interleukin-3 (II-3), interleukin-6 (IL-6), neural growth factor (NGF),VEGF, Granulocyte-Macrophage Growth Factor (GM-CSF), HGF, Hox family,and Notch In a preferred embodiment of the invention the cells arecultured in the absence of EGF.

Preferably the positive growth factors or combination of growth factorsused in the present invention are fibroblast growth factor (FGF) (e.g.FGF-4 and FGF-2), IL-3, stem cell factor (SCF), FLT3 ligand,thrombopoietin (TPO), granulocyte macrophage-colony stimulating factor(GM-CSF), and neural growth factor (NGF). In embodiments of theinvention, FGF (e.g. FGF-4 or FGF-2) is used with SCF and FLT3 ligand;FGF is used with TPO; or TPO is used with SCF and FLT3 ligand.

In an aspect of the invention the proliferation conditions involve usingFGF-4 or FGF-2, SCF and FLT3 ligand to prepare cellular compositions toproduce non-hematopoietic cells such as osteoclasts, osteoblasts, musclecells, endothelial cells, hepatocytes, astrocytes, neural cells, and/oradipocytes. In another aspect the proliferation conditions involve usingTPO, SCF and FLT-3 ligand to prepare cellular compositions to producenon-hematopoietic cells such as endothelial cells. In another aspect theproliferation conditions involve using NGF, SCF, and FLT-3 to preparecellular compositions to produce non-hematopoietic cells such asendothelial cells or others.

The growth factors may be used in combination with equal molar orgreater amounts of a glycosaminoglycan such as heparin sulfate.

Growth factors may be commercially available or can be produced byrecombinant DNA techniques and purified to various degrees. For example,growth factors are commercially available from several vendors such as,for example, Genzyme (Framingham, Mass.), Genentech (South SanFrancisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems(Minneapolis, Minn.) and Immunex (Seattle, Wash.). Some growth factorsmay be purified from culture media of cell lines by standard biochemicaltechniques. Thus, it is intended that molecules having similarbiological activity as wild-type or purified growth factors (e.g.,recombinantly produced or mutants thereof) are intended to be usedwithin the spirit and scope of the invention.

An effective amount of a positive growth factor is used in the culturemedium. Generally, the concentration of a positive growth factor in theculture medium is between 10 and 150 ng/ml, preferably 25 to 100 ng/ml.The growth factors are typically applied at sufficient intervals tomaintain high proliferation levels and maintenance of a stem cellphenotype. In an embodiment, the growth factors are applied about 2-4times per week, preferably 2-3 times per week.

The culture medium may comprise conditioned medium, non-conditionedmedium, or embryonic stem cell medium. Examples of suitable conditionedmedium include IMDM, DMEM, or αMEM, conditioned with embryonicfibroblast cells (e.g. human embryonic fibroblast cells or mouseembryonic fibroblast cells), or equivalent medium. Examples of suitablenon-conditioned medium include Iscove's Modified Delbecco's Medium(IMDM), DMEM or αMEM or equivalent medium. The culture medium maycomprise serum (e.g. bovine serum, fetal bovine serum, calf bovineserum, horse serum, human serum, or an artificial serum substitute [e.g.1% bovine serum albumin, 10 μg/ml bovine pancreatic insulin, 200 μg/mlhuman transferrin, 10⁻⁴M β-mercaptoethanol, 2 mM L-glutamine and 40μg/ml LDL (Low Density Lipoproteins)], or it may be serum free.

In an embodiment, the culture medium is serum free to provide cells withthe potential or increased potential to form non-hematopoietic cellsthat are free of serum proteins or biomolecules that may bind to thesurface of the cells. Cells cultured in such conditions may providenon-hematopoietic cells that have potential exposed novel antigenicsites. Such cells may be useful as immunogens. Thus, the inventionprovides a cellular composition or mitotic or differentiated cellstherefrom that are isolated and maintained in serum-free media.

The proliferation conditions entail culturing the enriched cellpreparation for a sufficient period of time so that cells in thepreparation develop potential or an increased potential to formnon-hematopoietic cells and tissues. The cells are generally maintainedso that the cells complete about 1-100 cell cycles, preferably 5-75 cellcycles; more preferably 2-50, 2-40 or 2-20, most preferably at leastabout 2-10 or 4-5 cell cycles. This will typically correspond to about 4to 40 days in culture, preferably about 2-20 days in culture, morepreferably at least or about 2-15 days or 4-10 days in culture, and mostpreferably at least about 4-8 days in culture.

The frequency of feeding the enriched hematopoietic cell preparation isselected to promote the survival and growth of cells with the potentialor increased potential to form non-hematopoietic cells. In an embodimentthe cells are fed once or twice a week. The cells may be fed byreplacing the entirety of the culture media with new media.

The cells in culture may be selected for hematopoietic stem andprogenitor cells (e.g. CD45⁺HLA-ABC⁺ cells) at a frequency to promotethe survival and growth of cells with the potential or increasedpotential to form non-hematopoietic cells. In a preferred embodiment ofa method of the invention, cells that are enriched for hematopoieticstem and progenitor cells (e.g. CD45⁺HLA-ABC⁺ cells) are reselected atintervals, preferably weekly, through positive or negative selectiontechniques known in the art and described herein.

The methods of the invention may be conducted on a large-scale, forexample a cellular composition of the invention may be isolated and/orexpanded in a bioreactors.

The method of the present invention leads to a newly created cellularcomposition comprising a population of cells with the potential orincreased potential to form hematopoietic and non-hematopoietic cells invitro and in vivo. The cells may have an altered differentiation programenabling the cells to form non-hematopoietic cells. The cells may havethe potential to differentiate into cells that exhibit morphological,physiological, functional and/or immunological features ofnon-hematopoietic cells. The cells may be further characterized byembryonic or early non-hematopoietic tissue markers (e.g. the earlymuscle marker Desmin).

In an aspect of the invention, an isolated and purified cellularcomposition is provided comprising or comprising essentially cellscharacterized by one or more of the following:

(a) CD45⁺HLA-ABC⁺

(b) Capable of differentiating into hematopoietic cells or hematopoieticprogenitor cells;

(c) Capable of differentiating into one or more, two or more, three ormore, four or more, five or more, or six or more, differentnon-hematopoietic cell types, including mesenchymal stem or progenitorcells, neural stem or progenitor cells, or endothelial stem orprogenitor cells; in particular, for example, endothelial cells,osteoclasts, osteoblasts, adipocytes, muscle cells, astocytes, andneural cells;

(d) round shape and non-adherent growth requirements;

(e) stem cell factor receptor (KIT)+

(f) FLT3 ligand receptor+;

(g) FGF receptor+;

(h) express embryonic stem cell proteins such as Oct4, Stage SpecificEmbryonic Antigen-3 (SSEA3), and/or Stage Specific Embryonic Antigen-4(SSEA4);

(i) HoxB4⁺;

(j) Flk-1⁺;

(k) CD34^(±);

(l) non-tumorigenic, i.e. the cells do not give rise to neoplasm ortumor or are free from neoplasia and cancer,

(m) CD38^(±); and

(n) derived from umbilical cord blood.

A cellular composition may comprise cells with the characteristics (a)and (c); (a), (b), and (c); (a), (b), (c) and (d); (a), (b), (c), (d)and (e); (a), (b), (c), (d), (e), (f), and (g); (a) through (e)inclusive; (a) through (f) inclusive; (a) through (g) inclusive; (a)through (h) inclusive; (a) through (i) inclusive; (a) through (j)inclusive; (a) through (j) inclusive, and (k); (a) through (j) inclusiveand (l) and (k); (a) through (j) inclusive, and (l) and (m); (a) through(j) inclusive, and (l), (m), and (n); or (a) through (i) inclusive and(k); (a) through (i) inclusive and (k) and (l); (a) through (i)inclusive and (k), (l), and (m); (a) through (i) inclusive and (k), (l),(m) and (n). In a preferred embodiment, an enriched cell preparation isprovided comprising cells with the characteristics (a), (b), (c) (d) and(e).

Cellular compositions of the invention may also be prepared usingpositive or negative selection techniques based on one or more of thecharacteristics of the cells of the composition as described herein.

Cells with the potential or increased potential to formnon-hematopoietic cells may be induced to differentiate into cells andtissues of non-hematopoietic lineages in vitro or in viva. These cellsmay also provide hematopoietic cells (e.g. stem and/or progenitorcells), preferably an expanded hematopoietic cell preparation.

The cells with potential or increased potential to formnon-hematopoietic cells may be induced to differentiate into cells ofnon-hematopoietic cell lineages, preferably cells that exhibitmorphological, physiological functional, and/or immunological featuresof non-hematopoietic cells. Cells from a differentiated cell preparationmay be characterized by expression of genetic markers ofnon-hematopoietic cell lineages (e.g. markers for muscle, neural,adipocyte, osteoclast, osteoblast, endothelial, astrocytes, pancreaticcells, retinal cells, renal cells, connective tissue cells, andhepatocytes), or physiological, immunological or functionalcharacteristics of cells of non-hematopoietic lineages. For example,non-hematopoietic cells can be screened for expression of tissuespecific markers such as Myo-D (muscle), FLK-1 (endothelial), glialfibrillary acidic protein (astrocytes), glucagon (islet-α cells),insulin (islet-β cells), somatostatin (islet-δ), pancreatic polypeptide(islet-PP cells), cytokeratins (CK), mucin MUC1, carbonic anyhydrase II,and carbohydrate antigen 19.1 (ductal cells), and NESTIN (neural).

In an aspect of the invention, the invention provides a method forproducing an isolated and purified cell preparation comprising musclecells, neural cells (neurons, astrocytes, type I and type II, andoligodendrocytes), adipocytes, osteoclasts, osteoblasts, endothelialcells, pancreatic cells (acinar, ductal, islet-α, islet-β, islet-δ, andislet-PP), kidney cells, retinal cells, corneal cells, connective tissuecells, or hepatocytes, using a cellular composition or method of theinvention.

Differentiated cells can be used to prepare a cDNA library relativelyuncontaminated with cDNA preferentially expressed in cells from otherlineages, and they can be used to prepare antibodies that are specificfor particular markers of non-hematopoietic cells.

In an embodiment, the cells in a cellular composition of the inventionmay be cultured in osteoclast differentiation medium (e.g. serumcontaining medium with GM-CSF) to differentiate the cells intofunctional osteoclasts. Osteoclasts may be identified by the ability ofthe cells to resorb calcium citrate substrate. The osteoclasts may beconverted into osteoblasts by culturing in osteoblast specificdifferentiation medium. Osteoblasts may also be produced by culturingcells or a cellular composition of the invention on osteoblastdifferentiation medium (e.g. a MEM with dexamethasone,glycerolphosphate, ascorbic acid, and serum). Osteoblasts may beidentified by expression of tissue specific markers such as CBRα.

Functional neural cells may be obtained by culturing cells of a cellularcomposition of the invention with a differentiation factor that inducesformation of neural cells such as neural growth factor. Neural cells maybe obtained by growing cells of a cellular composition of the inventionon media that induces differentiation of the cells to neural cells (e.g.DMEM medium with serum and retinoic acid). Neural cells may beidentified based on expression of neural specific markers such asneurofilament, NESTIN, and Parkin.

Muscle cells may be produced by culturing cells of a cellularcomposition of the invention with a differentiation factor that inducesformation of muscle cells. The cells of a cellular composition of theinvention may be cultured in specialized muscle specific cell culturemedia that may comprise a differentiation factor that inducesdifferentiation of the cells to form muscle cells. Muscle cells may beidentified by expression of mature muscle cell markers such as Myo-D andmuscle specific actin.

Endothelial cells may be produced by culturing cell of a cellularcomposition of the invention with a differentiation factor that inducesformation of endothelial cells. The cells of a cellular composition ofthe invention may be cultured in specialized endothelial cell culturesthat may comprise a differentiation factor that induces differentiationof the cells to form endothelial cells. Endothelial cells may beidentified based on expression of Flk-1 and/or CD31.

Hepatocytes may be produced by culturing cells of a cellular compositionof the invention with a differentiation factor that induces formation ofhepatocytes (e.g. n-butyrate). The cells of a cellular composition ofthe invention may be cultured in specialized cell cultures that maycomprise a differentiation factor that induces differentiation of thecells to form hepatocytes. Hepatocytes may be identified based onexpression of CYP1A2, alpha-fetoprotein, albumin, CK 19, and/or ICAM-1.

Astrocytes may be produced by culturing cells of a cellular compositionof the invention with a differentiation factor that induces formation ofastrocytes (e.g. G-5 astrocyte growth supplement). The cells of acellular composition of the invention may be cultured in specializedcell cultures that may comprise a differentiation factor that inducesdifferentiation of the cells to form astrocytes. Astrocytes may beidentified based on expression of glial fibrillary acidic protein(GFAP).

Adipocytes may be produced by culturing cells of a cellular compositionof the invention with a differentiation factor that induces formation ofadipocytes. The cells of a cellular composition of the invention may becultured in specialized cell cultures that may comprise adifferentiation factor that induces differentiation of the cells to formadipocytes. Adipocytes may be identified based on positive staining withSudanIV or oil-o-red.

Similarly, renal cells, retinal cells, corneal cells, and connectivetissue cells may be produced by culturing cells of a cellularcomposition of the invention with a differentiation factor that inducesformation of the cells, or they may be cultured in specialized cellcultures that induce differentiation to form the cells. The cells may beidentified based on expression of cell specific markers.

After differentiation of the cells into selected non-hematopoietic cellsas described herein, the cells may be separated to obtain a populationof cells largely consisting of the non-hematopoietic cells. This may beaccomplished by positive selection of non-hematopoietic cells usingantibodies to identify tissue specific cell surface markers or negativeselection using hematopoietic cell specific markers.

Expansion of hematopoietic stem cells and progenitor cells in accordancewith the invention can be carried out under proliferation conditions asdescribed herein. In general, the same culturing conditions that areused for culturing cells to produce cells with potential or increasedpotential to form non-hematopoietic cells may be employed. An exemplaryprotocol for expanding hematopoietic stem cells and progenitor cells isprovided in the Example.

Modification of Cells

A cell preparation or cellular composition of the invention may bederived from or comprised of cells that have been genetically modified(transduced or transfected) either in nature or by genetic engineeringtechniques in vivo or in vitro.

Cells in cell preparations and compositions of the invention can bemodified by introducing mutations into genes in the cells (or the cellsfrom which they are obtained) or by introducing transgenes into thecells. Insertion or deletion mutations may be introduced in a cell usingstandard techniques. A transgene may be introduced into cells viaconventional techniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, or microinjection. Suitable methods for transformingand transfecting cells can be found in Sambrook et al (2), and otherlaboratory textbooks. By way of example, a transgene may be introducedinto cells using an appropriate expression vector including but notlimited to cosmids, plasmids, or modified viruses (e.g. replicationdefective retroviruses, adenoviruses and adeno-associated viruses).Transfection is easily and efficiently obtained using standard methodsincluding culturing the cells on a monolayer of virus-producing cells(20, 21).

A gene encoding a selectable marker may be integrated into cells of acell preparation or composition of the invention. For example, a genewhich encodes a protein such as β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or a fluorescent protein markermay be integrated into the cells. Examples of fluorescent proteinmarkers are the Green Fluorescent Protein (GFP) from the jellyfish A.victoria, or a variant thereof that retains its fluorescent propertieswhen expressed invertebrate cells. (For example, the GFP variantsdescribed in references 22-24; and EGFP commercially available fromClontech Palo Alto, Calif.).

Another aspect of the present invention relates to geneticallyengineering the cells in the cell preparations and compositions of theinvention in such a manner that they or cells derived therefrom produce,in vitro or in vivo, polypeptides, hormones and proteins not normallyproduced in the cells in biologically significant amounts, or producedin small amounts but in situations in which regulatory expression wouldlead to a therapeutic benefit. For example, the cells could beengineered with a gene that expresses a molecule that specificallyinhibits bone resorption, but does not otherwise interfere withosteoclasts binding to bone, or the cells could be engineered with agene that expresses insulin at levels compatible with normal injecteddoses. Alternatively the cells could be modified such that a proteinnormally expressed will be expressed at much lower levels. Theseproducts would then be secreted into the surrounding media or purifiedfrom the cells. The cells formed in this way can serve as continuousshort term or long term production systems of the expressed substance.

Thus, in accordance with this aspect of the invention, cells with thepotential or increased potential to form non-hematopoietic cells can bemodified with genetic material of interest. The modified cells can becultured in vitro under suitable conditions so that they differentiateinto specific non-hematopoietic cells. The non-hematopoietic cells areable to express the product of the gene expression or secrete theexpression product. These modified cells can be administered to a targettissue where the expressed product will have a beneficial effect.

In a further embodiment, the transduced cells with the potential orincreased potential to form non-hematopoietic cells can be induced invivo to differentiate into non-hematopoietic cells that will express thegene product For example, the transduced cells may be administered toinduce production of non-hematopoietic cells having the transduced gene.The cells may be administered in admixture with each other or separatelyand maybe delivered to a targeted area. The cells can be introducedintravenously and home to the targeted area. Alternatively, the cellsmay be used alone and caused to differentiate in vivo.

Thus, genes can be introduced into cells which are then injected into arecipient where the expression of the gene will have a therapeuticeffect For example, osteoclasts may be genetically engineered to havereduced activity in viva Appropriate genes would include those that playa role in the regulation of osteoporosis, in areas such as serum calciumresponsiveness, estrogen secretion and bone resorption. An insulin genemay be introduced into blood stem cells to provide a constanttherapeutic dose of insulin in the bone marrow and peripheral blood.

The technology may be used to produce additional copies of essentialgenes to allow augmented expression by non-hematopoietic cells ofcertain gene products in viva. These genes can be, for example,hormones, matrix proteins, cell membrane proteins, cytokines, adhesionmolecules, or “rebuilding” proteins important in tissue repair.

Applications:

The cell preparations and compositions of the invention can be used in avariety of methods (e.g. transplantation) and they have numerous uses inthe field of medicine. They may be used for the replacement of bodytissues, organs, components or structures which are missing or damageddue to trauma, age, metabolic or toxic injury, disease, idiopathic loss,or any other cause.

Transplantation or grafting, as used herein, can include the steps ofisolating a cell preparation according to the invention and transferringcells in the preparation into a mammal or a patient. Transplantation caninvolve transferring the cells into a mammal or a patient by injectionof a cell suspension into the mammal or patient, surgical implantationof a cell mass into a tissue or organ of the mammal or patient, orperfusion of a tissue or organ with a cell suspension. The route oftransferring the cells may be determined by the requirement for thecells to reside in a particular tissue or organ and by the ability ofthe cells to find and be retained by the desired target tissue or organ.Where the transplanted cells are to reside in a particular location,they can be surgically placed into a tissue or organ or simply injectedinto the bloodstream if the cells have the capability to migrate to thedesired target organ.

The invention may be used for autografting (cells from an individual areused in the same individual), allografting cells (cells from oneindividual are used in another individual) and xenografting(transplantation from one species to another). Thus, the cells, cellpreparations and cellular compositions of the invention may be used inautologous or allogenic transplantation procedures to improve anon-hematopoietic cell or hematopoietic cell deficit or to repairtissue.

In an aspect of the invention, the newly created cellular compositionscomprising cells with potential or increased potential to formnon-hematopoietic cells, or non-hematopoietic cells differentiatedtherefrom, can be used in both cell therapies and gene therapies aimedat alleviating disorders and diseases involving the non-hematopoieticcells. The invention obviates the need for human tissue to be used invarious medical and research applications.

The cell therapy approach involves the use of transplantation of thenewly created cellular compositions comprising cells with the potentialor increased potential to form non-hematopoietic cells, ornon-hematopoietic cells differentiated therefrom, as a treatment forinjuries and diseases. The steps in this application include: (a)producing a cellular composition comprising cells with the potential orincreased potential to form non-hematopoietic cells, ornon-hematopoietic cells differentiated therefrom, as described herein;and (b) allowing the cells to form functional connections either beforeor after a step involving transplantation of the cells. The gene therapyapproach also involves cellular compositions comprising cells with thepotential or increased potential to form non-hematopoietic cells,however, following the culturing step in proliferation conditions, thenewly created cells are transfected with an appropriate vectorcontaining a cDNA for a desired protein, followed by a step where themodified cells are transplanted.

In either a cell or gene therapy approach, therefore, cells withpotential or increased potential to form non-hematopoietic cells andhematopoietic cells in cellular compositions of the present invention,or cells or tissues differentiated from the cells can be transplantedin, or grafted to, a patient in need. Thus, the cells with potential toform non-hematopoietic cells or differentiated cells therefrom can beused to replace non-hematopoietic cells in a patient in a cell therapyapproach, useful in the treatment of tissue injury, and diseases. Thesecells can be also used as vehicles for the delivery of specific geneproducts to a patient One example of how these newly created cells orcell differentiated therefrom can be used in a gene therapy method is intreating the effects of Parkinson's disease. For example, tyrosinehydrolase, a key enzyme in dopamine synthesis, may be delivered to apatient via the transplantation of a cell preparation of the inventioncomprising cells that are capable of differentiating into neuronalcells, or transplantation of neuronal cells differentiated from thecells, which have been transfected with a vector suitable for theexpression of tyrosine hydrolase.

The invention also provides a method of treating a patient with acondition involving a non-hematopoietic cell comprising transferring acellular composition comprising cells with the potential or increasedpotential to form non-hematopoietic cells into the patient, wherein thecell differentiates into the non-hematopoietic cells.

The invention provides a method for obtaining non-hematopoietic cellsfor autologous transplantation from a patient's own hematopoietic cellscomprising (a) obtaining a sample comprising hematopoietic cells fromthe patient, preferably from fresh or cryopreserved umbilical cordblood; (b) separating out an enriched cell preparation comprisinghematopoietic stem cells and hematopoietic progenitor cells, preferablyCD45⁺HLA-ABC⁺ cells; and (b) culturing the cells under proliferationconditions to produce a cellular composition comprising cells with thepotential or increased potential to form non-hematopoietic cells. Thecellular composition obtained from (b) can be cultured with adifferentiating factor, or cells of the composition can be transferredto the patient.

The invention also contemplates a pharmaceutical composition comprisingcells, a cell preparation, or cellular composition of the invention, anda pharmaceutically acceptable carrier, excipient, or diluent. Thepharmaceutical compositions herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective amount ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe cells, cell preparations, or cellular compositions in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffered solutions with a suitable pH and iso-osmotic withthe physiological fluids.

Still another aspect of the invention is a kit for producing cellularcompositions of the invention comprising cells that have potential orincreased potential to form cells capable of differentiating into cellsof multiple tissue types both in vitro and in vivo. The kit includes thereagents for a method of the present invention for producing a cellularcomposition. This kit preferably would include at least one positivegrowth factor, and instructions for use.

In an aspect, cells, cell preparations, and cellular compositionsdisclosed herein can be used for toxicity testing for drug developmenttesting. Toxicity testing may be conducted by culturing cells, cellpreparations, and cellular compositions or cells differentiatedtherefrom in a suitable medium and introducing a substance, such as apharmaceutical or chemical, to the culture. The cells or differentiatedcells are examined to determine if the substance has had an adverseeffect on the culture. Drug development testing may be done bydeveloping derivative cell lines which may be used to test the efficacyof new drugs. Affinity assays for new drugs may also be developed fromthe cells, differentiated cells, or cell lines.

Using a method of the invention it is possible to identify drugs thatare potentially toxic to non-hematopoietic or hematopoietic cells.

The cellular compositions of the invention may be used to screen forpotential therapeutics that modulate development or activity of cellswith the potential to form non-hematopoietic cells or cellsdifferentiated there from. In particular, the cells of a cellularcomposition of the invention may be subjected to a test substance, andthe effect of the test substance may be compared to a control (e.g. inthe absence of the substance) to determine if the test substancemodulates development or activity of the cells with the potential toform non-hematopoietic cells or cells differentiated therefrom.

In an aspect of the invention a method is provided for using cells withthe potential or increased potential to form non-hematopoietic cells orcells differentiated therefrom to assay the activity of a test substancecomprising the steps of

a) culturing cells in an enriched hematopoietic cell preparationcomprising hematopoietic stem cells and progenitor cells underproliferation conditions to obtain a cellular composition comprisingcells which have potential or increased potential to formnon-hematopoietic cells;

b) optionally culturing the cells which have potential or increasedpotential to form non-hematopoietic cells under differentiationconditions in vitro;

c) exposing the cultured cells in step (a) or (b) to a test substance;and

d) detecting the presence or absence of an effect of the test substanceon the survival of the cells or on a morphological functional, orphysiological characteristic and/or molecular biological property of thecells, whereby an effect altering cell survival, a morphological,functional, or physiological characteristic and/or a molecularbiological property of the cells indicates the activity of the testsubstance.

In another aspect a method is provide for using cells with the potentialor increased potential to form non-hematopoietic cells or cellsdifferentiated therefrom to screen a potential new drug to treat adisorder involving the non-hematopoietic cells comprising the steps of:

(a) obtaining hematopoietic cells from a sample from a patient with adisorder involving non-hematopoietic cells;

(b) preparing from the hematopoietic cells an enriched hematopoieticcell preparation comprising hematopoietic stem cells and progenitorcells;

(c) culturing the enriched hematopoietic cell preparation underproliferation conditions to obtain cells with potential or increasedpotential to form the non-hematopoietic cells;

(d) optionally culturing the cells with potential or increased potentialto form the non-hematopoietic cells under differentiation conditions invitro;

(e) exposing the cultured cells in (c) or (d) to a potential new drug;and

(f) detecting the presence or absence of an effect of the potential newdrug on the survival of the cells or on a morphological, functional, orphysiological characteristic and/or molecular biological property ofsaid cells, whereby an effect altering cell survival a morphological,functional, or physiological characteristic and/or a molecularbiological property of the cells indicates the activity of the potentialnew drug.

The invention also relates to the use of cells, cell preparations, andcellular compositions in drug discovery. The invention provides methodsfor drug development using the cells, cell preparations, and cellularcompositions of the invention. Cells, cell preparations, and cellularcompositions of the invention may comprise cells that secrete novel orknown biological molecules or components. In particular, culturing inthe absence of serum may provide cells that have minimal interferencefrom serum molecules and thus, may be more physiologically andtopologically-accurate. Therefore, proteins secreted by cells describedherein maybe used as targets for drug development. In one embodiment,drugs can be made to target specific proteins on cells that have thepotential or increased potential to form non-hematopoietic cells.Binding of the drug may promote differentiation of cells into specificnon-hematopoietic cells. In another embodiment, drugs specific forregulatory proteins of non-hematopoietic cells may be used to arrestgrowth of a particular type of cell. Any of the proteins can be used astargets to develop antibody, protein, antisense, aptamer, ribozymes, orsmall molecule drugs.

Agents, test substances, or drugs identified in accordance with a methodof the invention or used in a method of the invention include but arenot limited to proteins, peptides such as soluble peptides includingIg-tailed fusion peptides, members of random peptide libraries andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids, phosphopeptides (including members ofrandom or partially degenerate, directed phosphopeptide libraries),antibodies[e.g. polyclonal, monoclonal, humanized, anti-idiotypic,chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)2, and Fabexpression library fragments, and epitope-binding fragments thereof)],nucleic acids, ribozymes, carbohydrates, and small organic or inorganicmolecules. An agent, substance or drug may be an endogenousphysiological compound or it may be a natural or synthetic compound.

The cells, cell preparations, and cellular compositions disclosed hereincan be used in various bioassays. In an embodiment, the cells are usedto determine which biological factors are required for proliferation ordifferentiation. By using cells that have the potential or increasedpotential to form non-hematopoietic cells and hematopoietic cells in astepwise fashion in combination with different biological compounds(such as hormones, specific growth factors, etc.), one or more specificbiological compounds can be found to induce differentiation tonon-hematopoietic cells. Other uses in a bioassay for the cells aredifferential display (i.e. mRNA differential display) andprotein-protein interactions using secreted proteins from the cells.Protein-protein interactions can be determined with techniques such as ayeast two-hybrid system. Proteins from cells, cell preparations andcellular compositions of the invention can be used to identify otherunknown proteins or other cell types that interact with the cells. Theseunknown proteins may be one or more of the following: growth factors,hormones, enzymes, transcription factors, translational factors, andtumor suppressors. Bioassays involving cells, cell preparations, andcellular compositions of the invention, and the protein-proteininteractions these cells form and the effects of protein-protein orcell-cell contact may be used to determine how surrounding tissuecontributes to proliferation or differentiation of non-hematopoietic andhematopoietic cells.

In an aspect of the invention cells with potential or increasedpotential for forming non-hematopoietic cells obtained after culturing apreparation from cord blood stem cells maybe used to repair cell ortissue injury. They may also be used in the treatment of genetic defectsthat result in nonfunctional cells. The cord blood stem cells grown inproliferation medium may be transplanted directly to the site ofdefective cells in order to rescue the defect or delivered via the bloodstream by injecting the cells into the vein. In addition, gene therapyvectors may be integrated into the cord blood stem cells followed byengraftment of these engineered cells to their target tissues. Theintroduction of gene therapy vectors requires cell proliferation. Thesuccessful long term engraftment of the cells to the target tissuerequires they maintain a stem cell characteristic. High proliferationrates of cord stem cells has been achieved without differentiation,which should lead to successful gene therapy.

In an embodiment, hepatocytes obtained from differentiating cells of acellular composition of the invention, preferably derived from umbilicalcord blood, or precursor cells thereof, may be used to restore a degreeof liver function to a subject needing such therapy, perhaps due to anacute, chronic, or inherited impairment of liver function. Thus, theymay be used to treat liver disease or repair liver damage. Inparticular, hepatocytes obtained in accordance with the presentinvention may be used to treat a number of degenerative liver diseases.Non-functional liver cells where there is no apparent physical damagemay be treated through partial hepatectomy, followed by therapy usinghepatocytes obtained using the present invention. The hepatocytes may beencapsulated, or part of a bioartificial liver device.

In another embodiment, endothelial cells obtained from differentiatingcells of a cellular composition of the invention, preferably derivedfrom umbilical cord blood, or precursor cells thereof may be used forvascular repair and they can be used in cardiopulmonary bypass surgery.Endothelial cells may be transfected with genes which produce angiogenicfactors and used in gene therapy to stimulate angiogenesis in patientswith vascular or cardiac insufficiency.

In still another embodiment, muscle cells obtained from differentiatingcells of a cellular composition of the invention, preferably derivedfrom umbilical cord blood, or precursor cells thereof, may be employedto repair muscle, in particular striated or cardiac muscle. Thus, thepresent invention may be used to treat degenerative muscle disease. Thecells may be used in treating muscular dystrophy, cardiomyopathy,congestive heart failure; and myocardial infarction, for example.Genetic muscle disorders and cardiac muscle disorders may be treatedusing precursor muscle cells obtained using methods of the presentinvention. If muscle loss is due to the lack of neuronal connection(neuro-muscular disease), both the neural and muscle tissues can bereplaced using cells obtained using the present invention.

In a further embodiment, neural cells obtained from differentiatingcells of a cellular composition of the invention, preferably derivedfrom umbilical cord blood, or precursor cells thereof maybe used fortreating neurodegenerative disorders, a brain or spinal cord injury, orneurological deficit Neurodegenerative disorders which can be treatedinclude for example, Parkinson's disease, Huntington's disease, multiplesclerosis, Alzheimer's disease, Tay Sach's disease, lysosomal storagedisease, brain and/or spinal cord injury due to ischemia, stroke, headinjury, cerebral palsy, spinal cord and brain damage/injury, depression,epilepsy, schizophrenia, and ataxia and alcoholism.

Neural cells generated in accordance with a method of the invention maybe transfected with a vector that can express growth factors, growthfactor receptors, and peptide neurotransmitters, or express enzymesinvolved in the synthesis of neurotransmitters. These transfected cellsmay be transplanted into regions of neurodegeneration.

In a still further embodiment, bone or cartilage cells obtained fromdifferentiating cells of a cellular composition of the invention,preferably derived from umbilical cord blood, or precursor cellsthereof, may be used to repair bone, and in reconstructive surgery ordegenerative diseases. Artificial substrates or matrices can be used incombination with the cells to reconstitute tissues, implanted into thejoints of patients to replace or repair damaged or deficient cartilage.The cartilage cells may be useful in the treatment of diseases of thejoint, for example, osteoarthritis, inflammatory arthropathies, septicarthritis, and crystalline arthropathies, and they can be used toenhance healing of bone fractures when inserted into the site of afracture. The cells can also be used in the study and treatment ofchondrodysplasias, and to test angiogenic factors.

Retinal cells or precursor cells thereof generated in accordance with amethod of the invention may be used to restore vision lost when retinalcells are damaged, and they can be used as in vivo targets forstimulation by growth factors in order to produce healthy tissue. Inparticular the cells may be used to treat conditions such as glaucoma,macular degeneration, diabetic retinopathies, inherited retinaldegeneration such as retinitis pigmentosa, retinal detachment or injuryand retinopathies (whether inherited, induced by surgery, trauma, atoxic compound or agent, or, photically; in particular, diabeticretinopathy).

Connective tissue cells or precursor cells thereof, generated inaccordance with a method of the invention may be seeded onto matrices orsubstrates and used to repair or regenerate damaged tissue (e.g.tendons). Thus the invention contemplates a method for de novo formationof connective tissue in vivo by introducing connective tissue cellsproduced by a method of the invention into a site for de novo connectivetissue formation in a patient in need thereof.

Renal cells or precursors thereof, generated in accordance with a methodof the invention may be used to treat kidney disorders or damage, orrenal cancer. The cells, or tissue or a functioning kidney regeneratedtherefrom, may be administered to a patient to treat acute or chronicdecline in renal function. Functional renal cells or regenerated kidneycan be implanted into the donor of the hematopoietic cells from whichthe renal cells are derived or into another patient. Renal cells orprecursors there of may be used to construct an artificial kidney system(e.g. a system based on a hollow fiber filtration system.

Corneal cells or precursors thereof, generated in accordance with amethod of the invention may be used to treat a variety of corneal and/orconjunctival epithelial cell injuries, degenerations and/orabnormalities, including subjects having ocular surface diseases such asStevens-Johnson's Syndrome, chemical and thermal burns, ocular surfacetumors, immunological conditions, radiation injury, inherited syndromessuch as aniridia, ocular pemphigoid, macular degeneration, and the like.The corneal cells or precursors thereof may be particularly useful intreating patients where the normal stem cell population of the corneallimbus is depleted, non-functional or otherwise inadequate to promotehealing of the corneal damage.

The cells, cell preparations, and cellular compositions of the inventionmay be used as immunogens that are administered to a heterologousrecipient Administration of non-hematopoietic and hematopoietic cellsobtained in accordance with the invention may be accomplished by variousmethods. Methods of administering cells as immunogens to a heterologousrecipient include without limitation immunization, administration to amembrane by direct contact (e.g. by swabbing or scratch apparatus),administration to mucous membranes (e.g. by aerosol), and oraladministration. Immunization may be passive or active and may occur viadifferent routes including intraperitoneal injection, intradermalinjection, and local injection. The route and schedule of immunizationare in accordance with generally established conventional methods forantibody stimulation and production. Mammalian subjects, particularlymice, and antibody producing cells therefrom may be manipulated to serveas the basis for production of mammalian hybridoma cell lines.

The cellular compositions of the invention may be used to prepare modelsystems of disease. The cellular compositions of the invention can alsobe used to produce growth factors, hormones, etc.

In an aspect the invention provides a culture system from which genes,proteins, and other metabolites involved in proliferation ordifferentiation of hematopoietic or non-hematopoietic cells can beidentified and isolated. The cells in a culture system of the inventionmay be compared with other cells (e.g. differentiated cells) todetermine the mechanisms and compounds that stimulate production ofnon-hematopoietic and hematopoietic cells.

The cellular compositions of the invention can be used to screen forgenes expressed in or essential for differentiation of non-hematopoieticcells. Screening methods that can be used include RepresentationalDifference Analysis (RDA) or gene trapping with for example SA-lacZ(25). Gene trapping can be used to induce dominant mutations (e.g. bydeleting particular domains of the gene product) that affectdifferentiation or activity of non-hematopoietic cells and allow theidentification of genes expressed in or essential for differentiation ofthese cells.

The expanded cell preparations of the invention comprising increasednumbers of hematopoietic stem cells and progenitor cells may be used forenhancing the immune system of a patient. The cell preparations willfacilitate enhancement or reconstitution of the patient's immune and/orblood forming system.

In an aspect of the invention, the cellular compositions of theinvention are used in the treatment of leukemia (e.g. acute myelogenousleukemia, chronic myelogenous leukemia), lymphomas (e.g. non-Hodgkin'slymphoma), neuroblastoma, testicular cancer, multiple myeloma,melanomas, breast cancer, solid tumors that have a stem cell etiology,or other cancers in which therapy results in the depletion ofhematopoietic cells.

In another aspect of the invention, a cellular composition of theinvention, with or without genetic modification to provide resistance toHIV, is used to treat subjects infected with HIV-1 that have undergonesevere depletion of their hematopoietic cell compartment resulting in astate of immune deficiency.

The hematopoietic stem cells and progenitor cells in the expanded cellpreparation may also be transfected with a desired gene that can be usedfor treatment of genetic diseases. Hematopoietic cell-related geneticdiseases can be treated by grafting the expanded cell preparation withcells transfected with a gene that can make up for the deficiency or theabnormality of the gene causing the diseases. For example, a normal wildtype gene that causes a disease such as β-thalassemia (Mediterraneananemia), sickle cell anemia, ADA deficiency, recombinase deficiency,recombinase regulatory gene deficiency and the like, can be transferredinto the hematopoietic stem cells or progenitor cells by homologous orrandom recombination and the cells can be grafted into a patient.Further, a preparation comprising normal hematopoietic stem cells andprogenitor cells free from abnormalities of genes (from a suitabledonor) can be used for treatment.

Another application of gene therapy permits the use of a drug in a highconcentration, which is normally considered to be dangerous, byproviding drug resistance to normal hematopoietic stem cells bytransferring a drug resistant gene into the cells. In particular, it ispossible to carry out the treatment using an anticancer drug in highconcentration by transferring a gene having drug resistance against theanticancer drug, e.g., a multiple drug resistant gene into an expandedcell preparation comprising hematopoietic stem cells and progenitorcells.

Diseases other than those relating to the hematopoietic system can betreated by using the expanded cell preparations comprising hematopoieticstem cells and progenitor cells in so far as the diseases relate to adeficiency of secretory proteins such as hormones, enzymes, cytokines,growth factors and the like. A deficient protein can be induced andexpressed by transferring a gene encoding a target protein into thehematopoietic stem cells or progenitor cells under the control of asuitable promoter. The expression of the protein can be controlled toobtain the same activity as that obtained by the natural expression invivo.

It is also possible to insert a gene encoding a ribozyme, an antisensenucleic acid or the like or another suitable gene into the hematopoieticstem cells or progenitor cells to control expression of a specific geneproduct in the cells or to inhibit susceptibility to diseases. Forexample, the hematopoietic stem cells and progenitor cells can besubjected to gene modification to express an antisense nucleic acid or aribozyme, which can prevent growth of hematic pathogens such as HIV,HTLV-I, HTLV-II and the like in hematopoietic stem cells or cellsdifferentiated from hematopoietic stem cells.

The cell preparations comprising hematopoietic stem cells and progenitorcells can be introduced in a vertebrate, which is a recipient of cellgrafting, by, for example, conventional intravenous administration.

The invention also relates to a method for conducting a regenerativemedicine business, comprising: (a) a service for accepting and loggingin samples from a client comprising hematopoietic cells capable offorming cells that have the potential to form hematopoietic andnon-hematopoietic cells; (b) a system for culturing cells dissociatedfrom the samples, which system provides conditions for producing cellsthat have the potential to form hematopoietic and non-hematopoieticcells; (c) a cell preservation system for preserving cells generated bythe system in (b) for later retrieval on behalf of the client or a thirdparty. The method may further comprise a billing system for billing theclient or a medical insurance provider thereof.

The invention features a method for conducting a stem cell businesscomprising identifying agents which influence the proliferation,differentiation, or survival of cells that have the potential to formhematopoietic and non-hematopoietic cells. Examples of such agents aresmall molecules, antibodies, and extracellular proteins. Identifiedagents can be profiled and assessed for safety and efficacy in animals.In another aspect, the invention contemplates methods for influencingthe proliferation, differentiation, or survival of cells that have thepotential to form hematopoietic and non-hematopoietic cells bycontacting the cells with an agent or agents identified by the foregoingmethod. The identified agents can be formulated as a pharmaceuticalpreparation, and manufactured, marketed, and distributed for sale.

In an embodiment, the invention provides a method for conducting a stemcell business comprising (a) identifying one or more agents which affectthe proliferation, differentiation, function, or survival of cells thathave the potential to form hematopoietic and non-hematopoietic cells ofthe invention; (b) conducting therapeutic profiling of agents identifiedin (a); or analogs thereof for efficacy and toxicity in animals; and (c)formulating a pharmaceutical composition including one or more agentsidentified in (b) as having an acceptable therapeutic profile. Themethod may further comprise the step of establishing a distributionsystem for distributing the pharmaceutical preparation for sale. Themethod may also comprise establishing a sales group for marketing thepharmaceutical preparation. The invention also contemplates a method forconducting a drug discovery business comprising identifying factors thatinfluence the proliferation, differentiation, function, or survival ofcells that have the potential to form hematopoietic andnon-hematopoietic cells of the invention, and licensing the rights forfurther development.

Having now described the invention, the same will be more readilyunderstood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Example 1 Materials and Methods Maternal Blood Screening:

Maternal blood was screened for HIV I/II, HTLV-I/II, Hepatitis B (HBsAg), Hepatitis C (anti HVC), CMV and VDRL at the time of registrationprior to 34 weeks gestation. Written consent for collecting andprocessing umbilical cord blood was obtained at the time ofregistration. Qualified hospital personnel, following protocols approvedby the human ethics committee of the Toronto General Hospital and theUniversity of Toronto, collected the cord blood at the time of delivery.

Sample Processing:

The blood volume was reduced and the red blood cells removed with either

Ficoll or Pentaspan (starch) treatment Samples were collected using 60ml syringes containing the anticoagulant, Acid Citrate Dextrose (ACD) at1 ml per 10 ml of blood (10% v/v) or collected directly into a 250 mlblood bag (Baxter-Fenwal, Deerfield, II, USA) and Penicillin G was addeddirectly to the bag. Ficoll (Histopaque-1077, Sigma, St. Louis, USA)gradient centrifugation was used to obtain an enriched population ofmononuclear cells. Briefly, blood was diluted 1:1 with RPMI medium and30 ml was overlaid on a 15 ml cushion of Ficoll (1.077). The gradientwas centrifuged at 300 g for 30 min at room temperature and the layer ofmononuclear cells (MNC) was collected. The Ficoll layer below was alsocollected. The MNC and the Ficoll layer were both resuspended in 2×volume of wash solution (12.5 ml filtered plasma from the donor cordblood, 120 ml Iscoves modified Dulbecco medium, 3 ml ACD). The samplewas centrifuged at 300 g for 10 minutes at room temperature. Cellpellets were collected and combined.

Original volumes of the umbilical cord blood sample included the 10% v/vof ACD. Therefore a 100-ml volume included approximately 90 ml of bloodand 10 ml of ACD.

For starch processing, 1 ml of starch (Pentaspan, Dupont, Ill. U.S.A.)is added to 5 ml of blood, mixed, then centrifuged for 10 minutes at50.times.g. The leukocyte rich upper layer is collected and the cellsare pelleted by spinning at 400.times.g for 10 minutes. The pellet isresuspended in 5 ml of IMDM and 6 volumes of red cell lysis buffer(Ammonium Chloride Buffer). After 10 minutes at room temperature thecells are pelleted and washed 1× in PBS and either cryopreserved orresuspended in column buffer.

Cryopreservation:

All steps were performed on ice. The cell pellet was resuspended inIMDM/10% autologous serum or FBS/10% DMSO. Up to 6 aliquots were frozenper sample by placing the sample in Nalgene cryovials (Nalgenenunc,Rochester, N.Y.) in a −80° C. freezer overnight. For long term storage,samples were moved to liquid nitrogen (−196° C.).

Isolation of an Enriched Stem Cell or Progenitor Cell Population.

Different methods using two different columns were used for theisolation of stem or progenitor cells.

A. MACS column (Mitenyl Biotech., Germany). A positive selectionmagnetic column using a monoclonal antibody (Mab) to CD34. The MAb oncebound to the cell is bound to a metal bead and is subsequently retainedon the column, which is attached to a magnet. All of the other cells arewashed of the column. The column is removed from the magnet and theCD34+ cells are eluted.

B. Stem Sep column (Stem Cell Technologies) is a negative selectioncolumn and is better suited for the isolation of primitive stem cells.Since all stem cells may not be CD34+ (26) a negative selection columnremoves all known, unwanted cells, leaving behind an enriched stem cellpopulation. The antibody cocktail removes all mature lymphoid andmyeloid cells are removed as well as all late progenitor stage cells. Itcontains a set of lineage specific surface markers found on maturehematopoietic cells; CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66&GLYCOPHORIN A.

C. Cell Culture Systems:

A variety of cell culture systems were tested. A number of media weretested and the main components are as follows;

A. Conditioned medium: Human embryonic fibroblasts (HEF-CM) grown inαMEM with 20% FBS (fetal bovine serum) for 72 hours. The medium isremoved and the cells and debris is removed with centrifugation andfiltering (0.22 μM filter). The medium is stored at −20° C.

B. Non conditioned medium/serum free: IMDM, 1% bovine serum albumin, 10μg/ml bovine pancreatic insulin, 200 μg/ml human transferrin, 10-4Mβ-mercaptoethanol, 2 mM L-glutamine and 40 μg/ml LDL (Low DensityLipoproteins).

C. ES cell medium without LIF in the presence of mitomycin treated humanembryonic fibroblast cells.

Cells were grown in any one of the three media with or without thefollowing growth factors in any combinations; 25-100 ng/ml FGF-4, 25-100ng/ml FGF-2, 25-100 ng/ml IL-3, 25-100 ng/ml SCF, 25-100 ng/ml FLT3ligand, 25-100 ng/ml TPO, 25-100 ng/ml GM-CSF, 25-100 ng/ml IL-6, 25-100ng/ml NGF.

Cells were grown for 4 days to 76+ days in the medium with mediumchanges and growth factor changes either weekly, twice weekly, or thriceweekly. All cells were grown in NUNC brand tissue culture treated 6, 12or 24 well plates (Becton Dickinson, N.J., USA).

Flow Cytometry Analysis: Cell Number and Viability.

Samples were stained for various cell surface markers (Beckman-Coulter)and subjected to flow cytometer analysis; Coulter-Epics (Coulter.Burlington, Canada). Isotype controls were used in all cases. Allsamples were labeled for 10-20 minutes at 4° C., washed and fixed in 10%formalin, as per manufacturer's instructions.

Colony Forming Units Plating Assay

A pre-made methyl cellulose based colony assay medium (Stem CellTechnologies) was used. The medium is formatted to grow primitiveprogenitors (cat#H4435). Cells were plated at 500 CD34+ cells per 3 mlof medium or 1500-3000 cells per 3 ml of medium for non-selected cells.All populations were plated in duplicate and scored at day 12, 14, 16and 18.

LTC-IC Assays.

Cells were also grown in Long Term Culture-Initiating Cell assays thatallow for the growth of early hematopoietic progenitor cells. This assaylinks the CFU-assay and the NOD/SCID assay. Cells are grown on feedercells which allow for the long term growth of hematopoietic cells.

Column Re-Selection

For some experiments the cells were re-selected using the Stem Sepcolumn (Stem cell Technologies). Tissue culture cells were labeled withthe appropriate antibody cocktail and passed through the column asdescribed above. Output cells were deemed to be negative for the surfacemarkers targeted in the cocktail and these cells were either checked forpositive surface markers by flow cytometry, placed in CFU-assays,replated for continued tissue culture or used to engraft NOD/SCID mice.

NOD/SCID Mouse Engraftment

Non Obese Diabetic/Severe Combined Immune Deficient (NOD/SCID) mice wereused to test the engraftment potential of the human umbilical cord bloodstem cells. All experiments followed established protocols and receivedanimal ethics approval. NOD/SCID mice were maintained in a clean room inisolator racks and given food and water. The mice were irradiated 2hours before engraftment at 360 Rads using a Cs¹³⁷ source. The mice werethen injected by tail vein with 200 of cells. Mice were given an i.minjection of antibiotics and monitored daily. Survival rate was 80+% perexperiment. At 2 weeks, 4 weeks, or 6 weeks, animals were sacrificed bycervical dislocation. Femurs were removed and the bone marrow flushed.Cells were washed in PBS and the cell pellet was subjected to Red CellLysis Buffer for 3 minutes and washed again. Cells were eithercryopreserved for re-engraftment, analyzed by flow cytometry, orsubjected to RNA isolation for PCR analysis or DNA microarray analysis.

Liver, spleen, muscle and brain tissue were also isolated. Tissues weredivided into pieces and fixed in 4% paraformaldehyde followed byembedding in paraffin wax. Some of the tissue was separated into singlecell suspension using a mild trypsin treatment (27) and labeled withfluorescent antibodies as described above.

Immunohistochemistry and Immunocytochemistry

All tissues were fixed in 4% paraformaldehyde followed by washes in PBS.The tissue was dehydrated followed by embedding in paraffin wax. 6 μmsections were cut and placed on glass slides. Slides were dewaxed andsubjected to antibody staining with fluorescent tagged antibodies.Slides were analyzed on a deconvolution microscope.

Osteoclast:

Osteoclast formation was accomplished by culturing the cells in IMDM+10%serum+GM-CSF. Positive cell activity was determined by plating the cellson calcium citrate substrates and measuring loss of the substrate asactive osteoblasts absorb the bone like substrate.

TRAP Staining:

TRAP Staining was carried out as described in Minkin Cedric (28). Cellswere grown for various periods in either 96-well or 24-well plates underthe influence of several “factors”. Tartate resistant acid phosphatasestaining was carried out on cells as follows:

6.4 ml of naphthol-as-Bi-phosphoric acid (12.5 mg/ml indimethylformamide).

6.4 ml acetate solution (2.5 mol/L) pH=5.2

3.2 ml Tartrate solution (0.67 mol/L) pH=5.2

Fast Red TR (0.1 g)

64 ml of Distilled water

The above solutions were mixed and filtered. Cells were then incubatedfor 45 minutes at 37° C.

Resorption Studies:

Cells were grown for 3 weeks under conditions that are favorable forTRAP positive 10,000 cells were seeded on to osteologic slides and grownfor 2 weeks with media changes every two to three days. Media consistedof IMDM and GM-CSF with serum. At 10 and 14 days the experiment wasstopped and Von Kossa staining was carried-out.

Ultrastructural Studies:

Cord blood cells were grown under above conditions for 21 days in24-well plates. Cells were then scraped off and centrifuged in amicrotube at 600 g for 5 minutes. Re-suspension in 2% gluteraldehyde for1 hour followed by transfer to cacodylate buffer and subsequentprocessing for viewing under electron microscopy.

Endothelial;

Cells are grown in culture dishes or chamber slides as well as 3-Dcultures which allow for the formation of capillary networks. Forcultures on chamber slides, cells are plated in M119 medium with serum(10%), supplemented with endothelial growth factor supplement (Sigma).Cells are fed twice per week by the removal of medium without the lossof cells. Capillary formation is accomplished by placing 2,000-10,000cells in 5 μl volumes on a 0.5 ml matrix made by mixing 0.5 ml of 3mg/ml fibrinogen in M199 medium with 10 μl of thrombin (μg/ml) and thencovering the cells with a second 0.5 ml of matrix in a 24 well plate andcovering with 1 ml of M199+5% serum. Medium is changed once per week for3-4 weeks until endothelial cell networks develop.

Adipocytes:

Sudan IV staining was carried out to determine whether there is presenceof fats in the cells grown under the conditions described above. Brieflycells were fixed in 70% ethanol for 5 minutes followed by incubationwith a solution of Sudan IV (2 g/ml in 50:50 acetone:ethanol) foranother 5 minutes. Cells were then washed with 70% ethanol and thenviewed under a microscope.

Osteoblast:

α-MEM with 10-8M Dexamethasone, 10 mM β-glycerolphosphate, 0.2 mMAscorbic Acid; 10% Serum. Cells were grown for 3-4 weeks. For AlzirinRed staining, cells are fixed in 70% ice cold ethanol for 30 minutes,then stained for 10 minutes with 40 mM Alzirin red (pH 4.0).

Neural:

Cells were placed into DME+10% serum and grown for 3 weeks with twiceweekly media exchanges. Some cultures were supplemented with 10-50nanomolar Retinoic Acid.

Muscle:

Cells were grown in FGF, SCF, FLT3Ligand in Stem Span medium (Stem CellTechnologies) for 2-21 days. At any time point the cells were eithertransferred into 20% serum in DME (high glucose) for three weeks inorder to produce mesenchymal cells or transferred directly to variousmedium at 37° C. or 33° C. or 6% Oxygen (37° C.) in one of the fivelisted conditions:

A: αME+10% serum+50 μM 2-mercaptoethanol

B: αMEM+10% serum+50 μM 2-mercaptoethanol+5-Azacytidine

C: αMEM+10% serum+50 μM 2-mercaptoethanol+10 μg/ml insulin+0.1-1 μMDexamethasone+0.5 μM isomethylbutylxanthine

D: αMEM+10% serum+Chick embryo extract (5%)

E: αMEM+1% serum+Chick embryo extract (5%)

Cells were cultured for 2-4 weeks and tested for muscle specific markersby PCR and immunocytochemistry.

Hepatocytes:

Livers were isolated from mice that received cells through tail veininjections. Upon sacrifice, the livers were quickly removed and eitherfixed in 10% formalin and processed with paraffin wax forimmunohistochemistry or single cell suspensions were produced and cellswere stained with anti-HLA-ABC antibody and anti-CD45 antibody.

Results:

In all experiments listed below, cells were isolated from humanumbilical cord blood starting populations of cells were tested 1)unfractionated leukocytes 2) lineage minus cells (CD2, CD3, CD14, CD16,CD19, CD24, CD56, CD66 & GLYCOPHORIN A minus). Lineage minus cells areobtained by initially removing known mature blood cells thereby leavingbehind immature cells and cells lacking any known blood marker(unidentified cells). This population of lineage minus cells is HLA-ABCpositive, CD45⁺ (100%), and enriched for CD34 (50-80%), CD38 (50-80%),and CD33 (50%), a myeloid marker and 3) Lineage positive cells. Insummary, but detailed below, the key population is the lineage minuspopulation because in the majority of studies lineage positive cellsfailed to show any stem cell properties, and the frequency of cells withstem cell potential found in the unfractionated cell population suggeststhey are from the lineage minus population contained within.

The Appearance of Non-Blood Cells

Non-blood cells were produced from CD45⁺ UCB cells. First, it wasobserved that a combination of prolonged culturing with reduced feedingscaused the differentiation of these cells by letting them withdraw fromthe cell cycle. The cells begin to adhere resulting in mixed colonies ofelongated adherent cells and loosely attached to them are round cells.As long as FGF is present the cells can maintain these colonies, whichappear at about three weeks of culture. After three more weeks the roundcells begin to die and the adherent cells stop dividing but remainalive. The cells stained positive for the mesenchymal marker, vimentin.After 6-10 weeks in culture (total time) the adherent cells persist andshow morphologies resembling endothelial cells, adipocytes (fat), andosteoclasts. These specialized cells occur infrequently. By altering theculture conditions (as mentioned above and detailed below) the fate ofthe cells can be better controlled. Initial studies were performed inorder to determine the optimum cytokine type and concentration requiredto promote stem cell growth. The goal was to produce a proliferatingpopulation of cells that does not require serum, conditioned medium orfeeder cells in order to maintained multi-potential cell properties asdefined by the ability of the cells to give rise to mature hematopoieticand non-hematopoietic cells and tissues. It is important to eliminateboth the requirement for serum and conditioned medium as this is notfeasible for a clinical setting. Furthermore, reducing the dependence onserum and conditioned medium provides us with more control over themaintenance of cell phenotype and cell proliferation.

Mesenchymal-like cells can be directly obtained by plating whole cordblood directly into DME or IMDM plus 10-20% serum. (See belowmesenchymal cell intermediate).

Cytokine Supplementation:

Leukocytes from umbilical cord blood cells were depleted of all maturecells leaving an enriched stem and progenitor cell population. Thesecells can be maintained in serum free conditions with growth factorsupplementation for approximately three months, although the majority ofcell proliferation occurs in the first 28 days then tapers off. Culturescan be maintained in a highly proliferative state by feeding every 48hours and separating the lineage minus cells from the lineage positivepopulation as done at the beginning of the culture period every 7-10days or on a continuous basis. FGF-2 and FGF-4 were focused upon due totheir ability in the murine embryo to maintain proliferation ofnon-differentiated cells. Furthermore, FGF-2 and FGF-4 are downregulated prior to cells undergoing differentiation, thus making it anideal candidate for stem cell proliferation. FGF-2 and FGF-4 were testedin conjunction with +/− serum, +/− cytokines, on different substrates.The studies showed no difference between FGF-2 and FGF-4. However, FGF-4alone was used because of its use in maintaining human embryonic celllines. The addition of SCF and FLT-3ligand to the FGF-4 containingmedium increased the proliferation rate. FGF, SCF or Flt-3ligand usedalone in serum free conditions resulted in reduced cell proliferation,while combinations of these cytokines resulted in improved yields ofstem cells. SCF had a minor effect on cell proliferation rates whenadded to FGF, Flt-3ligand cultures, but was critical in blockingdifferentiation of the stem cell pool (FIG. 1).

The frequency of FGF supplementation, but not the concentration, greatlyaffected the outcome of the cultures. 100 ng/ml and 25 ng/ml of FGF-4 orFGF-2 were tested with 25 ng/ml of FLT-3 ligand and 25 ng/ml of SCF.There was no difference in the cell proliferation rate, the frequency ofCD34⁺/CD38⁻ cells or CFU's when the supplementation schedule was keptconstant. This is due to the rapid degradation rate of FGF. Once perweek feeding for 4 weeks resulted in maintenance of some cells with HSCcharacteristics but the majority of cells differentiated and died.Interestingly FGF-4, FLt-3L, SCF, serum free, with once/week mediachanges resulted in massive cell proliferation for three weeks, followedby the majority of cells dying with the remainder forming mixed coloniesof round, non-adherent and elongated adherent cells of equalproportions. Feedings of about 3 times per week are required to maintainhigh proliferation levels and maintenance of the stem cell phenotype.Furthermore, reselection of the cells at weekly intervals through thesame negative selection column helped to maintain and expand the lineageminus population. By supplementing the cultures with growth factorsthree times per week the cells were maintained as round, non-adherentcells. The cells have been maintained in this state for about 80 daysbefore terminating the experiment

Other Cytokines:

Although some cytokine supplementation protocols tested resulted inmassive proliferation the rate of differentiation was also highresulting in a decreased number of stem cells of the total cellpopulation. IL-3 with Flt-3 and SCF resulted in massive cellproliferation but the lineage minus population was lost as theydifferentiated rapidly. TPO with Flt-3 and SCF resulted in a betterbalance of cell proliferation and stem cell maintenance. NGF, SCF andFLT-3ligand also gave similar results to FGF. TPO, SCF & FLT3ligandcultured cells were also compared to FGF, SCF, FLT3 ligand cells.Although the TPO treated cells proliferate much better than FGF cells(12-20 fold increase vs. 4-10 fold), TPO cells have a more limited rangeof producing non-hematopoietic lineages.

Formation of Non-Hematopoietic Cells is not Dependent on a MesenchymalCell Intermediate.

The adherent cell population that appeared with infrequent feedings oras a result of growing unfractionated cord blood cells in serumconditions, is reminiscent of the mesenchymal cell population found inbone marrow aspirates. First it was investigated whether the adherentstage of growth observed in the original population was a mesenchymalcell population similar to that found in bone marrow, and 2) a mandatorystep towards non-blood differentiation. Jiang et al (29) reported aCD45⁻ (non-blood), mesenchymal cell population isolated from bone marrowthat is capable of producing a wide range of cell types from a clonalcell population. We tested 1) Lineage negative cells (stem andprogenitor cells), 2) Lineage positive cells (mature blood cells) and 3)unfractionated UCB cells were tested.

Lineage Negative Cells:

UBC Lin− cells were grown in SCF and FLT-3 ligand with or without FGF-4.After three weeks of growth, mixed colonies appear that consist ofstromal like cells and round hematopoietic cells. Cells grown in FGF-4,SCF, Flt3ligand, produce a greater number of these colonies.Furthermore, if the cells are fed at 2-3 feedings/week versus once/weekthe cultures contain more non-adherent single cells. These round,non-adherent cells are still capable of forming mesenchymal cells oncethe growth factors are reduced or the cells are placed into serumcontaining medium. Growing the cells in FGF, SCF and Flt3ligand allowsan increase in the number of cells with mesenchymal cell properties.Since the mesenchymal like cells appear after three weeks of growth, theround nonadherent cells that dominant the cultures in the first 2-3weeks were tested. The expanded, non-adherent cells contain the abilityto generate specific non-hematopoietic cell types (as described inMaterials and Methods and below). Therefore with frequent feedings thecells tend to remain non-adherent and retain their potential forproducing non-hematopoietic cells. Furthermore the round, non-adherentcells remain CD45⁺ and HLA-class I⁺ (FIG. 2).

If the UBC lin− cells were placed directly into conditions which promotethe growth of adherent, mesenchymal-like cells (DME+20% serum) the cellswould die after one week. When the lin⁻ cells were grown for 8 days inFGF-4, SCF, Flt3ligand prior to culture in serum medium, vimentinpositive cells would develop. These cells could be maintained in serumconditions for .about.6 weeks. Furthermore, these cells lost theirability to produce positive blood forming cells when tested in CFUassays and LTC-IC in vitro assays. These cells also died when placed incultures that promote endothelial cell development (VEGF containingcultures) or osteoclasts. Interestingly, FGF-4, SCF, Flt3ligand growncells placed into DME+10% serum would be 50% positive for the neuralmarker, neurofilament. Therefore conditions which lead to vimentinpositive cells also give rise to neural cells. For muscle and bone,placing the cells from FGF-4, SCF, Flt3ligand cultures, directly intogrowth conditions which cause tissue specific differentiation, wouldresult in positive cells for the specific tissue tested. UCB Lin− cellsplaced directly into bone or muscle differentiating medium would die. Anincrease in muscle and osteoblast precursor cells would occur when thecells were first grown in FGF-4, SCF, Flt3ligand medium and then DMEM orIMDM plus serum (10-20%) for 14 days. Stromal/Mesenchymal like cellshave also been identified from UCB in vivo. Lineage depleted UCB cells(UBC Lin−) were injected into NOD/SCID mice. Upon analysis of the bonemarrow of the mouse after 10 weeks, stromal and hematopoietic cellpopulations were identified. CD45 is a pan human leukocyte marker andHLA-ABC is a pan human cell marker. Cells that are HLA-ABC positive andCD45 negative are human, non-blood cells, and most likely stromal cells(FIG. 3).

Lineage Positive (Lin⁺):

These cells were grown in identical conditions as the lin− cells andtested for the presence of adherent cells as well as non-blood markers.The majority of lin⁺ cells died (95%) within 7-10 days of culture.Surviving cells produced in a minority of adherent cells ( 1/1,000,000cells) which failed to divide and died. Lineage positive cells grown intissue specific medium resulted in the death of all cells. Thisindicated that the cells removed (CD2, CD3, CD14, CD16, CD19, CD24,CD56, CD66& GLYCOPHORIN A positive cells) are not capable of producingnon-hematopoietic cells.

Non-Fractioned:

Cells were placed directly into 20% serum/DME or IMDM. The frequency ofadherent cells was 10/100,000. These cells proliferated slowly for 6weeks then died. Interestingly the best yield of adherent cells camewhen the non-adherent, non-fractioned cells cultured in DME orIMDW/10-20% serum for 4 days were transferred to a new culture chamberwith fresh medium. Within 24 hours of transfer, 10% of the cells adhere.The few non-adherent cells left in the original well died. After 46weeks in culture the number of live adherent cells was similar to thenumber adherent cells from the Lin− cell fraction grown for the samelength of time, strongly suggesting that the active, key cell is foundin the lineage negative population. The adherent, non-fractionated cellsare vimentin positive and have identical properties to that of the Lin−adherent cells.

Lineage+ and unfractioned cells were also grown in FSF cultures for 7days prior to the above tests. No differences in the results wereobserved when compared to ngA pre-culturing in FGF-4, SCF, Flt3ligandmedium. Any effect on the lineage negative cells contained within theunfractionated cells would be masked by the large number of non-reactivelineage positive cells. Therefore, F FGF-4, SCF, Flt3ligand growthperiod has a unique effect on Lineage minus cells.

It was previously reported that NGF-receptor is expressed on mesenchymaland some blood cells in the bone marrow. Isolation of NGF-R⁺ cells fromthe bone marrow is enriched for mesenchymal cells capable of developinginto osteoblasts and fibroblasts (30). FACS-sorting of a NGF-R⁺population from human UCB did not result in cells with mesenchymalproperties. Cells were isolated and grown in conditions that areconducive to mesenchymal cell growth. The positive cells did not survivewell in these cultures and never adhered as expected. Although nottested, the cells are probably the blood cell NGFR positive population.

How Early do the UBC Cells Begin to Display Non-Hematopoietic Markers?

The observation that UCB cells grown in serum conditions can give riseto vimentin positive cells and cells that morphologically resemblenon-blood types prompted testing of UBC cells at different; stages ofgrowth for the expression of non blood markers. Day 0 lin− cells werenegative for all non-blood markers tested. Since adherent cells do notappear until day 21 of culture in FSF medium, the ability ofnon-adherent, early stage cells at day 2, 4, 8, 14 and 21 were tested.In these cultures after at least 4 days (one-two cell divisions),non-hematopoietic embryonic and early tissue specific markers arise.

Lineage negative and positive cells as well as non-selected cells weretested. Cells were either moved directly into tissue-specific in vivoand in vitro assays that enabled determination of their developmentalpotential for blood and non-blood cells, or the cells were firstcultured under conditions which, depending on the growth factor used,may allow for an increased capacity of the CD45+ population to grow intonon-blood cells.

A combination of PCR analysis, antibody staining, enzymatic assays andin vivo functional assays was used to test the non-hematopoieticpotential of UCB cells. Day 0 Lin⁻ cells are negative for non-bloodmarkers but 100% positive for HLA-ABC and the blood marker CD45.Differentiation of cells into non-blood cells was a two-stage process.The first stage required growing UCB lin⁻ cells in FGF-4, SCF,Flt3ligand for a minimum of 4 days or in 10-20% serum in DMEM or IMDM asoutlined above.

PCR analysis and protein detection with antibodies to non-blood markersshowed that day 0 lin− cells are negative for nestin (neural), Desmin,Cardiomyosin (muscle), GFAP (astrocyte) FLK-1 (mesoderm andendothelial), CBFα-1 (bone) and Oct4 (embryonic stem cell) by PCR. Thesame cells are negative by antibody for FLK-1, CD31 (endothelial),Oct-4, Neurofilament, Nestin, Parkin (neural), GFAP (astrocyte), Cyp1A2hepatocyte), CBFα-1 (osteoblast), Desmin, MyoD and muscle actin(muscle).

Cells were also negative for TRAP and calcium citrate substrateresorption (osteoclast), negative for calcium deposits by Aizirin redstaining (osteoblast) and Sudan IV staining (adipocytes). Although day 0lin− cells are enriched for CD34, a surrogate marker for hematopoieticstem cells, these observations indicate that there is no relationshipbetween CD34 and the appearance of multipotent stem cells. During theculture period in cytokine supplemented, serum free medium, CD34 cellsincrease in frequency along with the appearance of multipotential stemcells (UCB stem cells) but the UCB stem cells are also found in theCD34− population. The CD38 marker is lost within the first 4 days andCD33 appears during this time. By day 8 greater then 80% of the cellsare CD33+ but this exceeds the number of UCB stem cells.

Cells were grown for 2, 4, 8, 14 and 21 days in FGF-4, SCF, Flt3ligandfed 3.times./week. Day 2 cells remained negative for the same PCRmarkers while cells grown for 4 days or more expressed nestin, desmin,GFAP, Flk-1 and Oct-4. The presence of Oct-4 is highly significant as itis considered a marker of stem cells. To explore this further UBC lin−cells were grown in IL-3 with SCF and Flt-3 ligand, Neural Growth Factorwith SCF and Flt-3 ligand or TPO with SCF and Flt3-ligand. IL-3supplemented cultures grew rapidly but were negative for Oct-4, desmin,nestin. NGF supplemented cultures were positive for only OCT4.TPO-cultured cells at day 8 are negative for nestin and FLK-1, positivefor OCT4 and weak to negative for Desmin by PCR. Thus demonstrating areduced multipotency when compared to FGF cells.

This is an important observation because OCT4⁺ or FLK1⁺ cells areprobably an important intermediate cell type (e.g., all matureendothelial cells grown are direct derivatives of the FLK1⁺ population),OCT4 or FLK1 positive cells are not found in the starting population.Therefore, the cell population found in UCB capable of multipotency is aCD45⁺/HLA-ABC⁺/FLK1⁻/OCT4⁻ cell. Since UCB stem cells respond well andtheir appearance in the cultures are dependent on FGF, SCF and Flt-3ligand the key cells are also FLT3 receptor (member of the receptortyrosine kinase class III receptors), SCF receptor (c-Kit), and FGFRIIpositive.

After 2-4 days of growth the cells are positive for non-blood embryonicmarkers from a wide variety of tissues. In order to furtherdifferentiate these cells they had to be placed into tissue-specificcell cultures. The cells are capable of giving rise to multiple bloodand non-blood cell types.

In summary, although lineage minus cells in culture will give rise tolineage positive (mature blood) cells, they also give rise to a novelcell type, not found in the original population, that will producenon-blood lineages. These cells seem to be a product of cultureconditions as untreated, day 0 lineage minus cells are negative for allnon-blood indicators.

These cells could be further differentiated when placed into specificculture conditions that promote the production/growth of a specific celltype (e.g. osteoclast). Therefore, the cells do not have to go throughan adherent phase and the production of vimentin positive cells is not arequired intermediate step but it is mandatory that the cells grown inserum free culture in order to allow for the development ofnon-hematopoietic lineages.

Blood

Despite the fact that that Fgf, Scf, Flt31 cells are able to expressnon-blood markers, they still maintain their ability to express bloodmarkers. The cells from the umbilical cord are collected in a mannerused to isolate cells capable of reconstituting the blood lineages.Growth and proliferation of a stem cell population capable of formingnon-blood cells should also maintain its ability to form blood cells.Hematopoietic stem cell maintenance and proliferation was tested usingin vitro and in vivo assays. Colony forming unit (CFU) assays, LTC-ICand cell surface marker analysis were used to indicate hematopoieticstem cells. In subsequent experiments NOD/SCID assays were used toverify stem cell phenotype and the ability of these cells to engraft thebone marrow.

Using the above serum free/conditioned medium-free media outlined abovethat resulted in an increase of cells expressing non-blood markers, anincrease in CFU's and LTC-IC was also observed over the first 8 days inculture. Surface analysis of cells grown in Fgf, SCF, Flt3ligand for 4-8days resulted in a shift in the population from a predominantly CD34⁺ toa CD33⁺ population. There was also an increase in CD34⁻, CD38⁻, andCD33⁻ cells. All cells maintained CD45⁺. Cells were maintained for up to80 days but in most cases there were few cells left to analyze. Cellsnever lost their CD45 marker and in some cases where there was CD45^(Io)or CD45⁻ cells in the initial population, these cells either died orturned on CD45 by day 8 as 100% of the population is positive (FIG. 4).

In vitro studies clearly show an increase in hematopoietic stem cellnumbers during an 8 day+ culture period. In order to address theengraftment potential of the in vitro expanded stem cells, freshlyisolated cord blood mononucleated cells, freshly isolated Lin− cells, orin vitro grown cells, were used to engraft irradiated NOD/SCID mice(FIG. 5). An increase in input cells resulted in an increase level ofengraftment Furthermore, an equal number of Lin− placed in culture for aminimum of 8 days had the same engraftment potential as their day 0counterparts suggesting that more NOD-SCID repopulating cells wereproduced over the 8 days.

Endothelial Cells, Bone (Osteoclast and Osteoblast), Adipocytes, Muscle,Astrocytes and Neural Cells from Cord Blood Cells:

The cells were plated and maintained (with constant splitting ofcultures) at very low densities, which allows single cells to beobserved. Stromal like cells (adherent, flat cells) were not observed inthe cultures until 3-4 weeks of growth. Round cells (individual cellsare pinpointed in a dish for repeated observation) were observedbecoming more adherent after 3 weeks of growth. The cells flatten outand their progeny produce both round and flat cells forming a mixedcolony. Furthermore, cells maintained in suspension for up to 12 weeksremained alive and non-adherent Aliquots that were removed at one weekintervals and allowed to settle and became adherent Conversely, adherentcells could be trypsinized and returned to suspension cultures andcontinue to grow as non-adherent cells.

Adherent cultures grown for up to 12 weeks in low growth factor serumfree medium resulted in cell morphologies reminiscent of fat cells,endothelial cells and osteoblast cells. These cells appeared at lowfrequencies with all three types appearing in single cultures. In orderto determine the identity of these cells the culture conditions wereoptimized to increase the yields of these cells to the point whereenough could be obtained for analysis. As mentioned above, cells grownfor as little as 4 days could be induced to express non-blood tissues.

A) Osteoclast:

Days 0 cord blood stem cells were tested for markers for osteoclasts(TRAP). All samples tested were negative for this osteoclast marker.Cord blood stem cells (lineage minus) grown for 7, 14, 21 and 28 days inFGF-4, SCF, and FLT-3L are highly positive for TRAP (50%+), andmultinucleated, both characteristics of osteoclasts (FIG. 6A). Themaximum number of TRAP positive cells appeared at day 21 (80%) andleveled off to day 28. In order to measure functionality of theosteoclast-like cells, cells were placed on calcium citrate substrateand measured for absorption of the substrate. Cells grown in FGF, SCF,FLT3ligand were not functional osteoclasts despite being TRAP positive.The cells had to be placed into an osteoclast differentiation medium(serum containing medium with GM-CSF) in order to differentiate theminto functional osteoclasts as observed by resorption of a calciumcitrate substrate (FIG. 6B). Therefore, the culture conditions stronglyinduced osteoclast precursor production.

B) Osteoblast:

Differentiation cultures were used to produce osteoblast cells.Osteoblast cells have been produced from UCB Lin− cells cultured withgrowth factors for at least 14 days [proliferation medium] and thenplaced in bone specific differentiation medium. The cells in theproliferation medium are negative for mature bone markers and morphologybut have the capability to complete the differentiation program to giverise to mature bone cells with characteristics indicative of osteoblastcells. Prolonged culture periods with increased or decreased amounts ofgrowth factors do not result in alkaline or mineralized cells. In orderto differentiate the osteoblasts into more mature bone cells the cellsmust be transferred to bone specific medium. Freshly isolated Lin− UCBcells placed into bone medium die without producing mature bone cells.These same cells cultured in Fgf, Scf, Flt3ligand medium for 7 days,then transferred to bone specific medium resulted in cells that arealkaline phosphatase positive. Furthermore, mineralization has beenobserved as the cells tested positive for Alizarin red staining.

C) Muscle:

UBC lin⁻ cells were grown for 7 days in either FGF plus SCF, FLT-3L inserum free medium. The cells divided rapidly and maintained the roundmorphology of hematopoietic cells. At the end of the 8 day cultureperiod, cells were tested for the embryonic/early muscle marker Desminby RT-PCR A positive signal was achieved (FIG. 7). Cells were alsotested for the mature muscle marker Myo-D, but remained negative. Cellsthat were grown in FGF placed into muscle specific cell culture mediatested positive for myo-D and muscle specific actin byimmunocytochemistry (FIG. 8).

D) Endothelial:

Flk-1 is a marker of mesoderm cells as well as hemangioblasts andendothelial cells. Endothelial precursors are FLK-1 positive and thismarker is lost as these cells mature into functional endothelial cells.

Day 0 cord blood stem cells are negative for flk-1. When placed into a3D culture system, which allows for the production of vessels, all day 0cells died. This is similar to the fate of the Lin⁻UBC cells placed intoother specialized mediums. This indicates that non-treated day Lin⁻UBCcells do not have endothelial potential. When Lin⁻/UCB cells were grownin SCF, FLT-3L or TPO, SCF, FLT-3L for a minimum of 4 days and thenplaced in specialized endothelial cell cultures, the cells developedinto endothelial cells.

UCB Lin⁻ cells were grown for 0, 7, 14, 21 and 28 days, and each cellpopulation was placed into tissue culture conditions specific for theformation and support of endothelial cells. Two different cultures wereused. The first supports the growth of 3-D vessels. Cells were testedfor the embryonic endothelial cell marker Flk-1 and for the matureendothelial marker CD31. There were no positive cells at day 0. Thenumber of endothelial cells increased when cells were cultured for 7-28days. FIG. 9A illustrates that the Flk-1 marker is present on the roundimmature cells and is lost as the cells take on the adherent, elongatedendothelial morphology characteristic of endothelial cells. UCB Lin⁻cells grown in FGF, SCF, FLT-3L for at least 7 days were capable offorming small vessels in vitro (FIG. 9 B-F). Hypoxia can induce theproduction of VEGF, which induces the production of FLK-1 positiveendothelial cells. Hypoxia plus FGF-4 gave the highest percentage ofFLK-1 positive cells. Cells were harvested and stained for CD31expression (FIG. 10). 80% of the cells in endothelial culture for aminimum of 14 days were CD31⁺. Interestingly, cells must be seeded athigh density. After 7 days in cultures cells along the peripheryelongate and move outward. After 4-6 weeks in culture a network ofvessels is observed. The cells at the centre of the cell mass die offleaving an outer rim of vessels.

All of the experiments were repeated with cells sorted for FLK⁺ cells,CD34⁺ and CD45⁺. Only Flk1⁺ cells (+/− other markers) gave rise toendothelial cells.

E) Hepatocyte:

The ability of human UBC/Lin⁻ cells to produce functional liver cellswas tested. The cells were tested in an in vivo model due to the lack ofgood in vitro hepatocyte models. UBC/Lin⁻ cells either untreated orgrown in FGF for 7 days were injected via the tail vein into NOD/SCIDmice. After 6-10 weeks the livers were isolated, and a single cellsuspension was obtained. Mice that were positive for human blood cellengraftment had liver cells that were HLA-ABC⁺/CD45⁻ suggesting thecells are human, non-blood cells ( 5/25). These cells were isolated byFACS sorting of single cell suspensions of the livers and tested forCYP1A2 expression. Of this sub group (⅖) few were positive forfunctional liver cells as assessed by CYP1A2 positive expression. Thecells were stained with the anti-CD45 antibody, which is specific tohuman blood cells and anti-HLA-ABC, which is specific to all humancells. CD45 negative-HLA-ABC positive cells were identified in the mouseliver (FIG. 11A: arrow). Pre data suggests that the FGF-4 treated cellscontributed 3 times as many cells as non-treated cells. This indirectapproach allows identification of only cells in the liver that are humancells but not blood cells.

In order to identify functional human liver cells in the mice cells thatwere CYP1A2 positive were identified. CYP1A2 is an enzyme found in humanliver tissue. It is only induced in mouse livers treated with dioxins,thus eliminating the possibility of cross-reaction with the antibodyused in the assay. Furthermore the antibody used is specific to thehuman CYP1A2 and will not react with the murine CYP1A2 protein. NOD/SCIDmice were not treated with any liver damaging chemicals (such as carbontetra chloride) in order to maintain functionality of the newlyengrafted cells. Furthermore, it was preferred that new cells infiltrateand take up residence in a liver that is not dramatically damaged. Thisallows assessment of the ability of UCB cells to be used in livertherapies for genetically defective livers where no physical damage mayoccur. This also provides a non-surgical method of treating inbornerrors of metabolism. Therefore, only low levels of liver engraftmentwere expected About 10-20% of the CD45−/HLA-ABC⁺ cells detected in themouse liver are also CYP1A2 positive. This suggest that althoughengraftment levels can be high, there are fewer functional hepatocytes(FIG. 11B, C). The same livers were sectioned and immunohistochemistrywith CYP1A2 antibody detected positive cells (FIG. 12).

Freshly isolated UCB cells (MNC or Lin⁻) were negative for CYP1A2.UCB/Lin⁻ cells that were treated with FSF for 7 days prior to tail veininjection into NOD/SCD mice were also negative for CYP1A2 expressingcells.

E) Astrocytes:

Due to the stromal nature of the cells, the cells were tested for theastrocyte marker Glial fibrillary acidic protein (GFAP). As for theabove experiments, UBC Lin− cells were grown in serum free medium withgrowth factors for 0-7 days. The cells were then tested by PCR for theGFAP mRNA. Day 0 cells were negative, while cells grown for 7 days werepositive (FIG. 13). Cells were also tested for GFAP protein expressionby immunocytochemistry. Cells placed into medium supplemented with G-5astrocyte growth supplement, used to promote astrocyte growth, were alsopositive for GFAP.

F) Neural:

Human UCB lin⁻ cells were grown in proliferation medium (gfg Scf,Flt3ligand) for 0-7 clays and tested for the early neural marker NESTIN.UBC/Lin− cells at day 0 tested negative for nestin. Once cells weregrown for 7 days they became positive by PCR (FIG. 14). Since thesecells were showing neural stem cell potential the cells were grown invarious medium in order to induce the expression of mature neuralmarkers as well as neural morphologies. It was expected that at somepoint the cells might take on a neural sphere morphology. In order toinduce neural spheres the cells were grown inFGF/EGF/heparin/DMEM/F12HAM-S medium, as published for the growth ofneural stem cells (31), with and without serum. Day 0 and day 8 cellsdied after 3 days in the serum free medium, while cells survived inserum positive cultures they failed to form neural spheres. As for theexperiments listed above, day zero cells in any of the neural culturesfailed to express any neural markers. In order to express any neuralmarkers or morphology the UBC/Lin⁻ cells had to first be grown in theproliferation medium described herein.

When day 7 UBC/Lin⁻ cells were placed into DMEM/10% serum culturesapproximately 50% of the cells died while the remainder became elongatedand adherent after 2-3 weeks in culture. These cells resembled afibroblast morphology. Prolonged culture resulted in some cells (about50%) of the adherent cells taking on a neural morphology. These cellswere tested for the expression of neural filament protein, Parkin,nestin and Neural Specific Enolase. Cells with neural morphology werepositive for at least one of each of these neural markers. Neurospheres,Parkin and Neuropositive cells are illustrated in FIG. 15.

Interestingly, selective growth/survival of neural cells resulted when10 μm Retinoic Acid (RA) was added to cells grown in proliferationculture for 7 days then placed into DMEM/10% serum until adherent cellswere present All other non-neural cells died as the RA containingcultures had more cell death present and half the number of cells versusthe non-RA cultures after 7 days. Furthermore 90+% of the RA cells werepositive for Neurofilament while only 50% of the non-RA were positive(FIG. 15). Cells in either medium survived for 12 weeks before celldivision stops and the cells die. This is most likely due to the cellsreaching a terminally differentiated state. If cells grown inproliferation medium for 7 days were placed directly into RA/DMEM/10%serum, cell clumping occurred. These cells remained alive, but do notresemble the tightly compacted neurospheres demonstrated previously(31). Cells could be maintained in RA cultures and the cells arepositive for neurofilament

In another experiment, the cells after 7 days of growth in Fgf, Scf,Flt3ligand were placed into cultures with Neural Growth Factor (NGF). Inthe presence of NGF and SCF and FLT-3 ligand the cells survive and some(<2%) neurofilament positive and Parlin positive cells were obtained.Thus NGF has the ability to convert cord blood stem cell into a neuralstem cell. In contrast, NGF, SCF, Flt-3L cells were negative for theendothelial marker FLK-1 marker, as expected.

G) Adipocyte:

Mouse bone marrow derived stromal cells are capable of formingosteoclast and adipocyte cells in the same cultures. UCB Lin⁻ cells wereplaced in proliferation cultures and tested at the various days foradipocytes. Cells grown for 7-28 days were positive for adipocytes atlow levels (<1%) when stained with SudanIV. Although less than 1% of thecells stained positive, this is significant as no cells were detected inthe same cultures that lacked the growth factor GM-CSF (FIG. 16).

H) Single or Multiple Stem Cells:

Combined percentages of positive cells for at least one tissue-specificmarker indicates that at minimum, single cells are expressing at leasttwo unrelated markers. This suggests that one cell can give rise to twoor more tissues. Growth of UBC/Lin⁻ cells in the proliferation mediumfor only 7 days results: in all cells being CD45⁺/HLA-ABC⁺/CD33⁺,suggesting the presence of a single, multipotent, clonal population thatis responsible for all observed cell types. In order to confirm this,single cells were placed into 96-well plates and grown in theproliferation medium. On average only 5/96 wells contained healthydividing cells after 14 days of growth. Only one well continued to growafter 21 days and continued for 10 weeks in total before becomingquiescent and dying. The experiment was repeated with 10 cells per well10/96 wells contained growing populations after 7 days.

Although specific tissues could not be tested for, the fact that 10% of10 cells/well cultures had growth properties similar to our bulkculture, suggested that the starting population contains cells havingdifferent proliferation rates and survival rates but a single cell isresponsible for the observed results. Multipotency may be dependent oncell-cell interaction and single cell plating may disrupt signalingpathways that are important to the survival of UCB multipotent cells.

Discussion:

A simple culture system is reported that allows for the production ofmultipotent stem cells derived from Umbilical Cord Blood. Theavailability of UCB and the ease of banking large numbers of sampleswill ensure the availability of HLA matched samples. Furthermore, thesimplified culture system will allow for the expanded use of cord bloodcells for tissue therapies beyond hematopoietic uses.

The mechanism by which the cells of the umbilical cord blood are capableof differentiating into non-hematopoietic cells could be due to: 1) thecells being naturally multipotent but their cell fate is determined bythe surrounding cells or the local environment; or 2) the cell fate hasbeen determined but the cells are reprogrammed when they are placed inan alternate environment (trans-differentiation).

Repopulating hematopoietic cells can be classified as progenitor cells(CD34⁺, CD38⁺/− and Lin⁻), which have limited renewal capacity, and stemcells (CD34⁺/−, CD38⁻, Lin⁻), which are contained within the progenitorpopulation and have a much greater capacity for self-renewal. In vitroexpansion of progenitor cells can lead to their proliferation asmeasured by colony assays, or FACS analysis, but limited, if any, longterm repopulation occurs during mouse bone marrow reconstitution studies(32). Furthermore, the autocrine reaction between stem cells, progenitorcells and accessory cells (all found within the UCB) makes it difficultto sort out whether any stem cell proliferation that does occur is dueto the direct effect of exogenous cytokines or the indirect effectmediated by non-stem cells present in the initial culture (33).Furthermore in vitro culture can result in the loss of specific cellsurface markers resulting in combinations of surface molecules not foundin the human body.

The methods of stem cell expansion of UCB, whether for hematopoietic ornon-hematopoietic tissues, is the same. Treatment of the UCB cellpopulation with any factor that stimulates the cell cycle leads to anincrease in the number of stem cells, which can give rise to bothhematopoietic and non-hematopoietic tissues/cells. The stem cells thatgive rise to non-hematopoietic tissues may be a rare population and aminimum growth period may be required in order for them to multiple todetectable levels. Alternatively, cell division may deregulate thehematopoietic stem cells increasing their stem cell potential so theydevelop characteristics similar to embryonic stem cells (ES cells).While not wishing to be bound to any theory, the data suggests thelatter.

Studies using mouse bone marrow cells have demonstrated that these cellshave the potential to become non-hematopoietic tissues. The ability ofcells to trans-differentiate becomes a powerful tool for tissue therapy.It was illustrated herein that human umbilical cord blood stem cells cangive rise, in vivo, and in vitro to some non-blood tissues. Here a2-step culture system is reported. UBC cells can be induced to developmultipotent embryonic stem cell characteristics if placed intospecialized proliferation medium, prior to exposure to specializeddifferentiation cultures (tissue specific). Freshly isolated UCB stemcells will not produce specialized cells. The cells have to bepre-cultured/grown in a proliferation culture in order to increase theirtissue potential. The pre-culture acts to increase cell division,probably disrupting normal gene regulation resulting in a ‘blank slate’phenotype. Cells grown for a minimum of one week are positive for anumber of non-blood markers as detected by PCR, enzyme analysis, FACS orimmunohistochemistry. Cells grown in proliferation medium are positivefor embryonic or early non-blood tissue markers, such as the earlymuscle marker Desmin, but negative for the mature marker Myo-D. As shownfor osteoclasts and endothelial cells, after growing in theproliferation medium for at least 7 days the cells can be furtherdifferentiated into mature and functional cell types by growing them inspecialized, differentiation medium. These are identical characteristicsto that of embryonic stem cells.

Growth Factors:

Growth Factors are involved either directly or indirectly in theproliferation, induction and patterning of tissue. Cell proliferation iscontrolled by extracellular signals (hormones, growth factors, andcytomines) during G1 phase of the cell cycle. The cells respond to thesesignals, both stimulatory and inhibitory by way of a distinct set ofserine/threonine kinases, termed cdk for cyclin dependent kinases due totheir association with short lived regulatory proteins referred to ascyclins. Four mammalian G1 cyclins have been characterized D1, D2, D3,and E. Each of the D cyclins is able to associate with one or morekinases cdk2, cdk4 and cdk 6 (34). Furthermore, the D cyclins seemunique as they respond directly to growth factor stimulation and less tonormal endogenous cell cycle signals (35). The critical response periodfor the D cyclins is in G1, at START, as defined in yeast. Past thispoint the cell is no longer dependent on growth factors to continue thecell cycle (36).

Although the transition from G1 to S can be induced by various growthfactors, stem cells whether neural or hematopoietic, reside mainly inGo, not G1 (37). CD34+ cells induced to enter G1 using cytokines wereless likely to contribute to the repopulation cohort when compared toCD34+ cells treated with cytokines but remaining in Go (38). This resultemphasizes the fact that entrance into the cell cycle can lead todifferentiation. The addition of growth promoters (e.g., FGF-4 or SCF(stem cell factor) & others) may prevent differentiation by keeping stemcells cycling. Additionally, both FGF and SCF have been implicated asdirect blockers of apoptosis (39,40). Fibroblast growth factor (FGF),epidermal growth factor (EGF) and activin, are potent growthstimulators, which can alter D cyclin levels and promote proliferation(41, 42). Withdrawal of growth factors leads to reduced cyclin levelsand differentiation. Thus, growth factors are regulators ofproliferation and differentiation.

Cells will respond to a variety of cytokines and studies show that somehave stronger mitogenic properties than others. In the Ladd et al.,study (43) SCF, Flt3, 11-3 and IL-6 all have the capacity to stimulatecell proliferation but only IL-3 had the ability to maintain highproliferation rates. Other studies have indicated that specificcytokines such as SCF, although they are not strong mitogens, canprevent differentiation. SCF has the ability to increase CFU numbersalone or synergistically with other Growth factors. In vitro SCF iscapable of maintaining a population (increased survival) of progenitorsbut did not cause cell proliferation. Used in combination with IL-3 orG-CSF, SCF had an additive effect on progenitor cell numbers over time(44). FLT13 ligand has been implicated in the maintenance of the CD34positive cell population in the presence of the strong mitogenicactivity of IL-3 (45). In contrast to SCF or FLT-3 ligand, it is clearthat other factors such as BMP4, retinoic acid or TGF-β are strongdifferentiation factors. The TGF-β family member, BMP, is important asinducers of cell differentiation. In the mouse embryo, BMP's areimportant in the initiation of neural differentiation. Although themechanism is not clearly understood, BMP's have a negative effect on thecell cycle resulting in longer cell cycle times and increased geneactivation resulting in differentiation. For example, BMP-6 inducesmesenchymal cells to differentiate into osteoblasts and in bone marrow,BMP-6 reduces the stromal derived levels of IL-6 (46). IL-3 causes anincrease in overall cell numbers but a decrease in stem/progenitor cellsas measured by CD34 surface markers, TNFa causes a decrease in thenumber of LTC-IC's (47) and TGF-β reduces mouse BM engraftment (48).Although IL-3 in some conditions has a positive effect on progenitorcell proliferation, IL-3 may inhibit the ability of HSC to home to thebone marrow. Thus an increase in cell numbers in vitro is notaccompanied by an increase in bone marrow engraftment (49).

Conclusion:

A proliferation system is described that allows for the development andsubsequent expansion of a human umbilical cord derived stem cell thathas the ability to give rise to hematopoietic and non-hematopoietictissues.

Example 2 Hematopoietic Stem Cell Expansion

The experiments discussed in this Example were designed to produce morehematopoietic tissues from a single UCB sample in order to obtain enoughcells to carry out successful bone marrow transplants on single adultsor multiple adult patients. It is important to eliminate both therequirement for serum and conditioned medium as this is not feasible fora clinical setting. Furthermore, reducing the dependence on serum andconditioned medium provides more control over maintenance of stem cellphenotype and cell proliferation. To this end the ability of variousgrowth factors to maintain UCB-HSC in the presence of conditioned mediumfrom a human embryonic fibroblast cell line (CM-HEF) were tested. Cellswere then tested in serum free/conditioned medium free media Stem cellmaintenance and proliferation was tested using colony forming unit (CFU)assays and cell surface marker analysis as an initial indicator of stemcell proliferation (FIG. 17) In subsequent experiments, LTC-IC andNOD/SCID assays were used to verify stem cell phenotype and the abilityof these cells to engraft (FIG. 5).

The serum free conditioned medium plus FGF-4 aided cell proliferationbut was not as proficient as serum+conditioned medium+FGF4. Cultureswhere also set up where the conditioned medium was eliminated, usingmedium with 10% serum and FGF-4. Two mediums (DMEM and IMDM) weretested. The addition of SCF to the FGF-4 containing medium increased theproliferation rate. The serum was removed from the conditioned medium byreplacing both with a combination of TPO, or FGF-4+/−IL-3, SCF, FLT-3ligand. Note that IL-3 may affect stem cell homing to the bone marrow.

Equivalent stem cell expansion results were obtained usingnon-conditioned, serum free IMDM with the addition of 25 ng/ml each ofTPO or FGF-4, +[SCF, and FLT-3 ligand] to that of serum+conditionedmedium. Feedings of 2-3 times per week are required to maintain highproliferation levels and maintenance of the stem cell phenotype.Addition of 100 ng/ml of each growth factor at the same frequency didnot have an effect.

Using the above serum free/conditioned medium-free media resulted in anincrease in CFUs and LTC-IC over the first 8 days over day 0 cells asanalyzed by LTC-IC, flow cytometry for CD34+/CD38− cells, Lineagedepletion (Lin−), and NOD/SCID mouse studies. Although overall cellnumbers increased when cells were allowed to grow for an additional 8days (day 8-16) the hematopoietic stem cell population was depleted.

While the present invention has been described with reference to what ispresently considered to be a preferred embodiment, it is to beunderstood that the invention is not limited to the disclosedembodiment. To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1. A method for producing a cellular composition comprising multipotentcells that express CD45 and HLA-ABC and are capable of differentiatinginto different types of non-hematopoietic cells comprising: (a)enriching hematopoietic cells to obtain an enriched hematopoieticCD45⁺HLA-ABC⁺ cell preparation; (b) culturing the enriched hematopoieticCD45⁺HLA-ABC⁺ cell preparation in a medium comprising FGF-4, SCF andFLT-3 ligand to provide said multipotent cells.
 2. A method as claimedin claim 1 wherein the hematopoietic cells are obtained from a sourceselected from the group consisting of blood, blood fractions, bonemarrow and umbilical cord blood.
 3. A method as claimed in claim 1wherein the enriched hematopoietic cell preparation is enriched inCD45⁺HLA-ABC⁺CD2⁻CD3⁻CD14⁻CD16⁻CD19⁻CD24⁻CD56⁻CD66⁻glycophorin A⁻ cells.4. A method as claimed in claim 1 which further comprises inducing themultipotent cells to differentiate into cells and tissues ofnon-hematopoietic lineages in vitro or in vivo.
 5. A cellularcomposition produced by a method of claim
 1. 6. A method of claim 1wherein positive selection is used to obtain an enriched hematopoieticCD45⁺HLA-ABC⁺ cell preparation.
 7. A method of claim 1 wherein negativeselection is used to obtain an enriched hematopoietic CD45⁺HLA-ABC⁺ cellpreparation.
 8. A method of claim 1 further comprising culturing themultipotent cells in the presence of a differentiation factor thatinduces differentiation of the multipotent cells into cells expressingglucagon, insulin, somatostatin and/or pancreatic polypeptide.
 9. Amethod of claim 1 further comprising culturing the multipotent cells inthe presence of a differentiation factor that induces differentiation ofthe multipotent cells into pancreatic cells.
 10. A method of claim 9wherein the pancreatic cells are selected from the group consisting ofacinar, ductal, islet-α, islet-β, islet-δ, and islet-PP cells.
 11. Amethod of claim 1 further comprising culturing the multipotent cells inthe presence of a differentiation factor that induces differentiation ofthe multipotent cells into muscle cells.
 12. An isolated and purifiedcellular composition comprising cells characterized by the following:(a) CD45⁺HLA-ABC⁺; (b) round shape and non-adherent growth requirements;and (c) stem cell factor receptor (KIT)+.
 13. An isolated and purifiedcellular composition of claim 12 wherein the cells are furthercharacterized by the following: (a) CD45⁺HLA-ABC⁺; (b) Capable ofdifferentiating into hematopoietic cells or progenitor cells; (c)Capable of differentiating into one or more, two or more, three or more,four or more, five or more, or six or more, different non-hematopoieticcell types; (d) round shape and non-adherent growth requirements; (e)Stem cell factor receptor (KIT)+ (f) FLT3ligand receptor+; (g) FGFreceptor+; (h) express embryonic stem cell proteins; (i) HoxB4⁺; (j)Flk-1⁺; (k) CD34^(±); (l) non-tumorigenic; (m) CD38^(±); and (n) derivedfrom umbilical cord blood.
 14. A method of identifying the presence ofcells of a cellular composition as claimed in claim 12 in a mixed cellpopulation comprising: exposing the cell population to an antibody orfragment thereof immunogenetically specific for markers (a) or (c) inclaim 12, the occurrence of the markers being indicative of the presenceof the cells in the cell population.
 15. A method for obtainingnon-hematopoietic cells for autologous transplantation from a patient'sown hematopoietic cells comprising (a) obtaining a sample comprisinghematopoietic cells from the patient; (b) separating out an enrichedcell preparation comprising CD45⁺HLA-ABC⁺ cells; (b) culturing the cellsin a medium comprising FGF-4, SCF and FLT-3 ligand to producemultipotent cells with the potential or increased potential to formnon-hematopoietic cells.
 16. An isolated and purified cellularcomposition comprising multipotent cells that express CD45, HLA-ABC andOCT-4 and are capable of differentiating into different types ofnon-hematopoietic cells.
 17. A pharmaceutical composition comprising acellular composition of claim 16, and a pharmaceutically acceptablecarrier, excipient, or diluent.
 18. A method of treating a patient witha condition involving non-hematopoietic cells comprising transferring acellular composition of claim 16 into the patient, wherein the cells inthe composition differentiate into the non-hematopoietic cells.
 19. Amethod of claim 18 wherein the non-hematopoietic cells are pancreaticcells and the condition is diabetes.
 20. An isolated and purifiedcellular composition comprising multipotent cells that express CD45,HLA-ABC, OCT-4, GFAP, desmin, nestin and Flk-1 and are capable ofdifferentiating into different types of non-hematopoietic cells.